Beta-2-glycoprotein 1 is an inhibitor of angiogenesis

ABSTRACT

The present disclosure provides a method of inhibiting angiogenesis within a tissue of interest by providing either intact or nicked β2-Glycoprotein 1 (βGP1) to cells associated with the tissue. The presence of β2GP1 inhibits angiogenesis within the tissue, in part by preventing neovascularization into the tissue. The disclosure also provides a method for treating tumors by providing β2GP1 to the tumor.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

REFERENCE TO A “Microfiche Appendix” Not Applicable BACKGROUND OF THEINVENTION

1. Field of the Invention

The present disclosure relates to methods and compositions forinhibiting angiogenesis.

2. Description of Related Art

β2-Glycoprotein 1 (β2GP1), also known as apolipoprotein H, is a 50 kDaplasma protein that is an abundant plasma glycoprotein found both freeand associated with lipoprotein (Polz et al., FEBS Letters 102:183-186,1979; Wurm, H., Int. J. Biochem. 16:511-515, 1984). Although the precisephysiological role of β2GP1 is not known, in vitro studies suggest thatit likely functions as a natural anticoagulant. β2GP1 has been shown toinhibit intrinsic pathway activation (Schousboe, I., Blood 66:1086-109,1985; Schousboe and Rasmussen, Throm. Haemostasis 73:798-804, 1995),tenase (Shi et al., Throm. Haemostasis 70:342-345, 1993), andprothrombinase activities (Nimpf et al., Biochim. Biophys. Acta884:142-149, 1986; Goldsmith et al., Brit. J. Heamatol. 87:548-554,1994) on the surface of activated platelets and synthetic phospholipidvesicles. β2GP1 also inhibits the activity of activated protein C onprocoagulant surfaces (Matsuda et al., American Journal of Hematology49:89-91, 1995; Mori et al., Throm. Haemostasis 75:49-55, 1996). Plasmalevels of β2GP1 have also been shown to fall during disseminatedintravascular coagulation (Brighton et al., Brit. J. Heamatol.93:185-194, 1996), which is consistent with the consumption of β2GP1 inthis thrombotic process.

β2GP1 inhibits ADP-induced platelet aggregation (Schousboe, I., Throm.Res. 19:225-237, 1980; Nimpf et al., Atherosclerosis 63:109-114, 1987)and participates in the etiology of several thrombolytic diseases (see,e.g., Brighton et al., Brit. J. Heamatol. 93:185-194, 1996; Asherson andCervera, J Invest Dermatol 100(1):21S-27S, 1993; Mackworth-Young, C.,Immunol. Today 11:60-65, 1990; Kandiah et al., Lupus 5:381-385, 1996).β2GP1 preferentially binds to surfaces bearing negatively chargedphospholipids. β2GP1 binds the negatively charged lipidsphosphatidylserine (PS) and cardiolipin (CL), and regulates thrombosisby its ability to compete for the assembly of coagulation factors onPS-expressing platelet and endothelial cell membranes.

β2GP1 binds endothelial cells with high affinity, particularlyendothelial cells that express acidic phospholipids (Ma et al., J. Biol.Chem. 275:15541-15548, 2000; Del Papa et al., J. Immunol. 160:5572-5578,1998; Del Papa et al., Arthritis Rhuem. 40:551-561, 1997; Del Papa etal., Clinical & Experimental Rheumatology 13:179-185, 1995), andundergoes specific proteolytic cleavage (Horbach et al., Throm.Haemostasis 81:87-95, 1999). Other important in vivo targets of β2GP1interaction are apoptotic or necrotic cells, where anionic phospholipidsnormally located in the inner side of the cell membrane becomesurface-exposed and serve as targets for β2GP1 binding (Balasubramanianand Schroit, J. Biol. Chem. 273:29272-29277, 1998; Balasubramanian etal., J. Biol. Chem. 272:31113-31117, 1997). Subsequent to binding, theprotein undergoes a conformational change (Wagenknecht and McIntyre,Throm. Haemostasis 69:361-365, 1993; Borchman et al., Clin. Exp.Immunol. 102:373-378, 1995; Lee et al., Biochim. Biophys. Acta1509:475-484, 2000) that is recognized by a specific lipid/β2GP1dependent receptor on the surface of phagocytes (Balasubramanian andSchroit, J. Biol. Chem. 273:29272-29277, 1998). Similarly, theassociation of β2GP1 with lipids can serve as antigens forantiphospholipid/β2GP1 autoantibodies (aPLAs) that are associated withsystemic lupus erythematosus and antiphospholipid syndrome (McNeil etal., Proc. Natl. Acad. Sci. (USA) 87:4120-4124, 1990; Bevers et al.,Throm. Haemostasis 66:629-632, 1991; Galli et al., Lancet 335:1544-1547,1990). Although the structural rearrangements that are important forlipid-dependent phagocyte recognition and the generation ofantiphospholipid antibodies are unknown, it is unequivocal that both theprotein and the target membranes undergo critical changes in theirconformation.

β2GP1 is a single-chain glycoprotein composed of 326 amino acid residuesand consists of four complement control protein (CCP) modules (domains Ithrough IV), as well as a distinct C-terminal domain V (Steinkasserer etal., Biochem. J. 277:387-391, 1991). From the crystal structure of β2GP1(Bouma et al., EMBO Journal 18:5166-5174, 1999; Schwarzenbacher et al.,EMBO Journal 18:6228-6239, 1999), it is known that the four CCP domainsexhibit an elliptically shaped β-sandwich structure comprised of severalantiparallel β-strands wrapped around a well-defined hydrophobic corecontaining one conserved tryptophan each. In contrast, domain V foldsinto a central β-spiral with two small helices and carries a distinctpositive charge in the proximity of a surface-exposed loop regioncomprising Trp316. Domain V carries the lipid binding region within thelysine-rich sequence motif (281CKNKEKKC288) and a hydrophobic loop(313LAFW316) important to membrane binding.

From experiments designed to identify the lipid-binding site of βGP1(now known to be domain V), Hunt and Krilis identified an inactive formof β2GP1 that does not bind lipids and as a result did not bindlipid/β2GP1 complex-specific phospholipid antibodies (Hunt and Krilis,J. Immunol. 152:653-659, 1994; Hunt et al., Proc. Natl. Acad. Sci. (USA)90:2141-2145, 1993). Sequence analyses of the inactive form yielded twoN-terminal sequences. One of the N-terminal sequences corresponded tothe N-terminus of the intact active form of β2GP1, while the other was anew sequence that started at Thr-318. The authors concluded that theinactive form was cleaved in domain V between amino acids Lys-317 andThr-318 but was still a single polypeptide because the disulfidesdelineating the five domains remained intact. Although the crystalstructure of the nicked protein has yet to be determined, it isreasonable to assume that the cleavage of β2GP1 at Lys 317/Thr 318results in a dramatic change in the proteins conformation since itslipid binding properties are essentially abrogated and lipid/βGP1complex-specific antiphospholipid antibodies no longer bind the protein.Using the crystal structure of the intact protein, molecular modeling,and epitope mapping with monoclonal β2GP1 antibody, Matssura et al.(International Immunology 12:1183-1192, 2000) proposed that cleavageresults in novel hydrophobic and electrostatic interactions in domain Vthat affect lipid and phospholipid antibody binding. Importantly, thesechanges might propagate down through the polypeptide to other domains.

Varying levels of endogenous nicked protein can be found in the plasmaof certain individuals, especially leukemia patients (Itoh et al., J.Biochem. 128:1017-1024, 2000) and patients treated with streptokinase(Horbach et al., Throm. Haemostasis 81:87-95, 1999). In vitro, βGP1 isproteolytically cleaved by enzymes that participate in the coagulationcascade; factor Xa, elastase, and plasmin all clip β2GP1 at Lys-317 andThr-318. Moreover, the addition of urokinase to plasmin inhibitor (α2PI)depleted plasma also generated nicked β2GP1 (Ohkura et al., Blood91:4173-4179, 1998). These observations suggest that activation offibrinolysis in vivo induces the cleavage of intact β2GP1 by plasminwhich results in the generation of the nicked protein (Blank et al.,Proc. Natl. Acad. Sci. (USA) 96:5164-5168, 1999). These data takentogether with the observations that β2GP1 binds endothelial cells(George et al., Circulation 99:2227-2230, 1999; George et al.,Circulation 97:900-906, 1998) through annexin II (Ma et al., J. Biol.Chem. 275:15541-15548, 2000) raises the possibility that localizedproduction of plasmin at the endothelial invasion front during woundrepair could cleaveβ2GP1. Thus, the nicked form of β2GP1 could begenerated directly on the cell surface, which would result in alteredproperties that directly affect endothelial cell function.

Angiogenesis, the process by which new blood vessels are formed bysprouting from pre-existing vessels, is a highly regulated process thatinvolves endothelial cell proliferation, proteolysis of matrixmolecules, self-association, elongation and migration. Angiogenic agentsmay be used to induce angiogenesis, or it may be the result of a naturalcondition. Angiogenesis is essential to a variety of normal activities,such as reproduction, development, tissue and organ growth, and woundrepair, and involves a complex interplay of molecules that stimulate andinhibit the growth and migration of endothelial cells, the primary cellsof the capillary blood vessels. Normally these molecules maintain themicrovasculature in a quiescent state (i.e., without capillary growth)for prolonged periods which can last for several years or even decades.

While angiogenesis is essential to homeostasis (Adams et al., Genes &Development 13:295-306, 1999; Erlebacher et al., Cell 80:371-378, 1995),its timely inhibition is critical to normal wound healing anddevelopment. For example, during wound repair endothelial cells canundergo rapid proliferation, with a much shorter turnover time (Folkmanand Shing, J. Biol. Chem., 267:10931-34, 1992; Folkman and Klagsbrun,Science 235:442-47, 1987). Should the balance between pro andantiangiogenic regulators go awry, uncontrolled capillary formation suchas that seen in rheumatoid arthritis, diabetic retinopathy, psoriasis,retrolental fibroplasias, hemangiomas, and tumor cell growth, as well asmetastasis, may result (Folkman, J., Nature Medicine 1:27-31, 1995;Ellis and Fidler, European Journal of Cancer 32A:2451-2460, 1996).

There are many diseases, categorized as “angiogenic diseases,” which arecharacterized by persistent unregulated angiogenesis. Unregulatedangiogenesis can either be the direct cause of the particular disease,or it may exacerbate an existing pathological condition. For example,ocular neovascularization appears to be the most common cause ofblindness and underlies the pathology of several eye diseases. Inarthritis, newly formed capillary blood vessels can invade joints anddestroy cartilage. In diabetes, new capillaries may form in the retinaand invade the vitreous humor, which can cause bleeding and blindness.

The growth and metastasis of solid tumors can also beangiogenesis-dependent (Folkman, J., Cancer Res. 46:467-73, 1986;Folkman et al., “Tumor Angiogenesis,” Chapter 10, pp. 206-32, in TheMolecular Basis of Cancer, Mendelsohn et al., eds. (W. B. Saunders,1995)). Tumor cells must attract new vessels to expand locally andproduce metastasis. For example, tumors that enlarge to greater than 2mm. in diameter need to obtain their own blood supply, and can do so byinducing the growth of new capillary blood vessels. These new bloodvessels become embedded in the tumor and provide nutrients and growthfactors essential for tumor growth, as well as a means for tumor cellsto enter the circulation and metastasize to other distant sites (Weidneret al., New Eng. J. Med., 324(1):1-8, 1991). Drugs that function asnatural inhibitors of angiogenesis have been shown to prevent the growthof small tumors in tumor-bearing animals (O'Reilly et al., Cell79:315-328, 1994). Sometimes the use of such negative regulators leadsto tumor regression and dormancy even after cessation of treatment(O'Reilly et al., Cell 88:277-85, 1997). Additionally, it has also beenshown that supplying inhibitors of angiogenesis to certain tumors canpotentiate their response to other therapeutic regimens (e.g.,chemotherapy).

During the past decade, several negative regulators of angiogenesis havebeen discovered. Compounds that have been reported to inhibitendothelial cell proliferation in different experimental systems includeTGF-β, (Muller et al., Proc. Natl. Acad. Sci. (USA) 84:5600-5604, 1987),thrombospondin (Good et al., Proc. Natl. Acad. Sci. (USA) 87:6624-6628,1990), IL-1 (Cozzolino et al., Proc. Natl. Acad. Sci. (USA)87:6487-6491, 1990), IFN-γ and IFN-α (Friesel et al., J. Cell. Biol.104:689-696, 1987), tissue inhibitor of metalloproteinase-1 (TIMP-1)(Takigawa et al., Biochem. Biophy. Res. Commun. 171:1264-1271, 1990),platelet factoi 4 (PF4) (Maione et al., Science 247:77-79, 1990),protamine (Taylor and Folkman, Nature 297:307-312, 1982), fumagillin(Ingber et al., Nature 348:555-557, 1990) and angiostatin (O'Reilly etal., Cell 79:315-328, 1994).

Some of these proteins are proteolytic fragments of the same proteinsthat control the balance between the formation and dissolution of fibrinclots formed as a result of tissue damage and wound healing. Forexample, proteolysis of antithrombin III (van Boven and Lane, Seminarsin Hematology 34:188-204, 1997), thrombin (Tsopanoglou et al., AmericanJournal of Physiology 264:C1302-C1307, 1993), and plasminogen, which allplay a critical role in controlling clot formation and angiogenesis,result in the production of cleaved antithrombin III (O'Reilly et al.,Science 285:1926-1928, 1999; Larsson, et al., J. Biol. Chem.276:11996-12002, 2001), prothrombin fragments 0.1 and 2 (Rhim et al.,Biochem. Bioph. Res. Co. 252:513-516, 1998), and angiostatin (O'Reillyet al., Cell 79:315-328, 1994; O'Reilly et al., Nature Medicine2:689-692, 1996), respectively, all of which inhibit the growth ofvascular endothelial cells. Although several angiogenesis inhibitors arecurrently under development for use in treating diseases, there exists aneed for better therapeutic options for inhibiting angiogenesis.

BRIEF SUMMARY OF THE INVENTION

The present disclosure describes methods for inhibiting, treating, orpreventing unwanted endothelial cell proliferation, cord formation, cellmigration, angiogenesis and angioectasia, especially as related tocancer or tumor growth, by the administration of an angiogenesisinhibitor, Beta-2-glycoprotein 1 (β2GP1). β2GP1 plays an importantregulatory role in endothelial cell physiology, angiogenesis, and tumorbiology, and has important therapeutic implications in inhibiting theangiogenic properties of proliferating tumors. In particular, β2GP1 isable to abrogate angiogenesis and angioectasia, inhibit endothelial cellgrowth, cord formation, and cell migration, and significantly inhibittumor growth and metastasis. Also disclosed are methods of inhibiting,treating, or preventing unwanted endothelial cell proliferation and/ormigration, as well as angiogenesis and angioectasia within a tissue ororganism by providing β2GP1 to cells associated with the tissue ororganism. Inhibition of angiogenesis and/or angioectasia is useful forthe treatment of diseases and conditions associated with increased orabnormal angiogenesis and/or endothelial cell proliferation.

As used herein, the term “β2GP1” includes both the intact form of β2GP1and the nicked form of β2GP1 (“N-β2GP1”). The amino acid sequence ofβ2GP1 varies slightly between species, and such variations fall withinthe scope of the term β2GP1. As used herein, N-β2GP1 is a β2GP1 proteinthat is cleaved at Lys 317/Thr 318. In a preferred embodiment, theN-β2GP1 is still a single polypeptide. As used herein, the term “β2GP1polypeptide” includes polypeptides, proteins, and peptides of β2GP1. Itis to be understood that β2GP1 polypeptides, proteins, peptides,analogs, derivatives, and fragments thereof may have endothelial and/orangiogenesis inhibiting activity and that any such β2GP1 polypeptides,proteins, peptides, analogs, derivatives, and fragments thereof fallwithin the scope and spirit of the present disclosure.

The present disclosure provides methods for treating diseases andprocesses mediated by undesired and uncontrolled angiogenesis and/orangioectasia by administering to a cell, a tissue, or an organism acomposition comprising a substantially purified β2GP1 in a dosagesufficient to inhibit, prevent, or treat angiogenesis and/orangioectasia. The present disclosure is particularly useful for treatingor for repressing the growth of neoplasms or tumors, as well as reducingtumor mass. Administration of β2GP1 to a human or animal withprevascularized metastasized tumors can prevent the growth or expansionof those tumors. The presence of β2GP1 inhibits, prevents, or treatsangiogenesis and/or angioectasia within a tissue or organism, in part bypreventing or arresting neovascularization into the tissue and bloodvessel dilation within a tissue. β2GP1 may be provided to a cell,tissue, or organism exogenously, by upregulating the expression ofendogenous β2GP1, or by exogenously providing an expression vector orconstruct that expresses β2GP1 in the target cell, tissue, or organism.

The disclosure also includes diagnostic methods and kits for determiningthe prognosis of an organism by assaying for the presence of β2GP1 inbiological fluids such as plasma and/or within a cancer or tumor. Thediagnostic methods and kits can be in any configuration well known tothose of ordinary skill in the art. The present disclosure also includesantibodies specific for β2GP1 and antibodies that inhibit the binding ofantibodies specific for β2GP1. These antibodies can be polyclonalantibodies or monoclonal antibodies. The antibodies specific for β2GP1can be used in diagnostic kits to detect the presence and quantity ofβ2GP1, which may be diagnostic or prognostic for the occurrence orrecurrence of cancer or other disease mediated by angiogenesis.

The methods of the present disclosure are clinically useful for treatinga host of diseases and conditions associated with angiogenesis, and forinterfering with angiogenesis associated with reproductive functions.Diseases and conditions that are mediated by angiogenesis include, butare not limited to, hemangioma, neoplasm, cancer, solid tumors,leukemia, metastasis, angioectasia, telangiectasia, psoriasis,scleroderma, pyogenic granuloma, Myocardial angiogenesis, plaqueneovascularization, cororany collaterals, ischemic limb angiogenesis,corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, arthritis, diabetic neovascularization, maculardegeneration, wound healing, peptic ulcer, fractures, keloids,vasculogenesis, hematopoiesis, ovulation, menstruation, andplacentation. The methods are also diagnostically useful for assessingthe prognosis of tumors and other disorders associated withangiogenesis. Furthermore, the methods are useful reagents forinvestigation of angiogenesis in the laboratory setting.

A preferred embodiment of the present disclosure is a method ofinhibiting angiogenesis and/or angioectasia within a tissue comprisingadministering an effective amount of β2GP1 to cells associated with thetissue, wherein the amount is sufficient to inhibit angiogenesis withinthe tissue. Preferably angiogenesis and/or angioectasia is inhibited inhuman tissue. In preferred embodiments, the β2GP1 is intact β2GP1,N-β2GP1, or recombinant β2GP1. Preferably the β2GP1 is administered tothe cells by exposing a composition comprising β2GP1 polypeptide to thecells. In a preferred embodiment, the cells associated with the tissueare endothelial cells. Preferably the endothelial cells are selectedfrom the group consisting of pulmonary endothelial cells, heartendothelial cells, gastrointestinal endothelial cells, brain endothelialcells, lymphatic endothelial cells, genital-urinary endothelial cells,skin endothelial cells, bone endothelial cells, muscle endothelialcells, breast endothelial cells, retinal endothelial cells, endocrineendothelial cells, central nervous system endothelial cells, hepaticendothelial cells, and umbilical cord endothelial cells. In anotherpreferred embodiment, the tissue is a tumor, and the administration ofβ2GP1 to the tumor tissue inhibits neovascularization into the tumor. Inyet another preferred embodiment, an antiangiogenic agent isadministered to the cells in conjunction with β2GP1. Preferably theantiangiogenic agent is selected from the group consisting ofangiostatin, endostatin, trastuzumab, thrombospondin, IFN-α, TIMP-1,PF4, fumagillin, and mixtures thereof. In another preferred embodiment,the β2GP1 is supplied to the cells topically, intravenously,subcutaneously, by direct injection into the tissue, orintraperitoneally.

Another preferred embodiment of the present disclosure is a method ofinhibiting endothelial cell proliferation comprising administering aneffective amount of β2GP1 to the cells, wherein the amount is sufficientto inhibit endothelial cell proliferation. Preferably endothelial cellproliferation is inhibited in human endothelial cells. In preferredembodiments, the β2GP1 is intact β2GP1, N-β2GP1, or recombinant β2GP1.In other preferred embodiments, administering an effective amount ofβ2GP1 is also able to inhibit endothelial cell migration and/ordifferentiation, in particular inhibit endothelial cells fromdifferentiating into tubular capillary structures.

Yet another preferred embodiment of the present disclosure is a methodof inhibiting angiogenesis and/or angioectasia within a neoplasmcomprising administering an effective amount of β2GP1 to the neoplasm,wherein the amount is sufficient to inhibit angiogenesis and/orangioectasia within the neoplasm. Preferably angiogenesis and/orangioectasia is inhibited in a human neoplasm. In preferred embodiments,the β2GP1 is intact β2GP1, N-β2GP1, or recombinant β2GP1. Preferably theneoplasm is a tumor. In another preferred embodiment, administering aneffective amount of β2GP1 is also able to inhibit neovascularizationinto the tumor. In yet another preferred embodiment, administering of aneffective amount of β2GP1 is able to inhibit metastasis of the neoplasmor tumor. In yet another preferred embodiment, an antiangiogenic agentis administered to the neoplasm in conjunction with β2GP1. Preferablythe antiangiogenic agent is selected from the group consisting ofangiostatin, endostatin, trastuzumab, thrombospondin, IFN-α, TIMP-1,PF4, fumagillin, and mixtures thereof. In yet another preferredembodiment, a therapeutic agent useful in the treatment of the neoplasmis administered in conjunction with β2GP1. Preferably the therapeuticagent is selected from the group consisting of cisplatin, doxorubicin,paclitaxel, vincristine, and vinblastin.

A preferred embodiment of the present disclosure is a method ofinhibiting angiogenesis and/or angioectasia at a tumor site in a subjectcomprising administering an effective amount of β2GP1 to the subject,wherein the amount is sufficient to inhibit angiogenesis and/orangioectasia at the tumor site. Preferably the subject is human. Inpreferred embodiments, the β2GP1 is intact β2GP1, N-β2GP1, orrecombinant β2GP1. In other preferred embodiments, the route ofadministration of β2GP1 to the subject is oral, intravenous,intramuscular, intrathecal, intradermal, intraperitoneal, subcutaneous,intrapleural, intrauterine, rectal, vaginal, topical, intratumor,transdermal, or transmucosal.

Another preferred embodiment of the present disclosure is a method ofinhibiting angiogenesis and/or angioectasia at a site in a subjectsuffering from an angiogenic disease comprising administering aneffective amount of β2GP1 to the subject, wherein the amount issufficient to inhibit angiogenesis and/or angioectasia at the site.Preferably the subject is human. In preferred embodiments, the β2GP1 isintact β2GP1, N-β2GP1, or recombinant β2GP1. Preferably the β2GP1 isadministered to the subject orally, intravenously, subcutaneously,intramuscularly, or topically. In yet another preferred embodiment theangiogenic disease is characterized by persistent unregulatedangiogenesis. Preferably the angiogenic disease is selected from thegroup consisting of diabetic retinopathy, retrolental fibroplasia,trachoma, neovascular glaucoma, psoriases, immune-inflammation,non-immune inflammation, atherosclerosis, and excessive wound repair. Inother preferred embodiments, the site in the subject is dermis,epidermis, endometrium, retina, surgical wound, gastrointestinal tract,umbilical cord, liver, kidney, reproductive system, lymphoid system,central nervous system, breast tissue, urinary tract, circulatorysystem, bone, muscle, or respiratory tract.

Examples of other preferred embodiments of the present disclosureinclude, but are not limited to a method of inhibiting angiogenesisand/or angioectasia in melanoma tissue comprising administering fromabout 50 to about 200 mg of β2GP1 topically; a method of inhibitingangiogenesis and/or angioectasia in fibrosarcoma tissue comprisingadministering from about 200 to about 300 mg of β2GP1 intravenously; amethod of inhibiting angiogenesis and/or angioectasia in renal cellcarcinoma tissue comprising administering from about 25 to about 125 mgof β2GP1 intravenously; a method of inhibiting angiogenesis and/orangioectasia in breast cancer tissue comprising administering from about250 to about 400 mg of β2GP1 to the tissue by direct injection; a methodof inhibiting angiogenesis and/or angioectasia in prostate cancer tissuecomprising administering from about 150 to about 300 mg of β2GP1 to thetissue by direct injection; a method of inhibiting angiogenesis and/orangioectasia in bladder cancer tissue comprising administering fromabout 200 to about 300 mg of β2GP1 intravenously; and a method ofinhibiting angiogenesis and/or angioectasia in colon cancer tissuecomprising administering from about 50 to about 250 mg of β2GP1intravenously.

A preferred embodiment of the present disclosure is a pharmaceuticalcomposition comprising β2GP1 and a second antiangiogenic agent usefulfor the inhibition of angiogenesis and/or angioectasia. Preferably thesecond antiangiogenic agent is selected from the group consisting ofangiostatin, endostatin, trastuzumab, thrombospondin, IFN-α, TIMP-1,PF4, and fumagillin. Another preferred embodiment is a pharmaceuticalcomposition comprising β2GP1 and a therapeutic agent useful for thetreatment of a neoplasm. Preferably the therapeutic agent is selectedfrom the group consisting of cisplatin, doxorubicin, paclitaxel,vincristine, and vinblastin.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Antiangiogenic property of β2GP1: Control gel foam plugscontaining human serum albumin (A) and plugs containing intact β2GP1 (B)and N-β2GP1 (C) were implanted subcutaneously. The mice were sacrificedtwo weeks later. Note the dramatic vascularization of plug A and theclear non-adherent and non-vascularized plugs B and C, whichdemonstrates the ability of ⊖2GP1 to inhibit or preventneovascularization.

FIG. 2. Quantitative vascular volume of gel foam implants as describedin FIG. 1: Calculated vascular volumes appear under each set ofindividual plugs. Estimated μL vascular volume in the plugs wascalculated by comparing the counts of 10 pt of tail vein blood from miceinjected with ⁵¹Cr-labeled syngeneic red blood cells (cpm/μL) with cpmassociated with the gel foam implants. Plugs A, B, and C contained humanserum albumin, intact β2GP1, and N-β2GP1, respectively. Again, thevascular volume in B and C plugs was dramatically less than in A plugs,demonstrating the ability of β2GP1 to inhibit or preventneovascularization.

FIG. 3. Left Panel. MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) of pulmonary microvascular cells grown for 4 daysin the presence of the indicated proteins. Double dilutions from 100μg/mL. Middle Panel. Photomicrographs of typical cultures (same plate asthe MIT assay data); a: N-β2GP1; b: human serum albumin; c: intactβ2GP1; and d: control cells without any treatment. As shown, there is asubstantial reduction in the number of viable endothelial cells afterincubation with intact β2GP1 and N-β2GP1 compared to the controls. RightPanel. MTT assay of B16 melanoma cells (20×10³) grown for 5 days in thepresence of the indicated compounds. Double dilutions from 100 μg/mL. Asshown, neither N-β2GP1 or intact β2GP1 inhibited proliferation of thistumor cell line.

FIG. 4. C3H mice were injected with 10⁶ UV 2237 cells. Dailysubcutaneous injections with buffer alone, intact β2GP1, and N-β2GP1were started five days later for 14 days. After injecting the mice with⁵¹Cr-labeled red blood cells, the mice were sacrificed, tumors weighed,and tumor blood vessel volume was calculated. As shown, mice treatedwith intact β2GP1 and N-β2GP1 had a significant reduction in both tumorweight and blood capillary volume of the tumors.

FIG. 5. C57BL/6 mice were injected with B16F10 melanoma cellsintravenously (A) or subcutaneously (B). Daily intraperitonealinjections with buffer alone, intact β2GP1, or N-β2GP1 started 1 daylater and lasted for 3 weeks. (A) Mice were sacrificed, lungs removedand fixed; tumors were then enumerated. (B) Tumor size was measured atthe indicated time points. Mice treated with N-β2GP1 had a significantreduction in both the size of the subcutaneous growing tumors and thenumber of lung metastasis.

FIG. 6. Ability of β2GP1 to inhibit the formation of capillary-liketubes in in vitro Matrigel assays. Human umbilical cord vascularendothelial cells (HUVEC) (10⁵/mL) resuspended in medium M-200containing intact β2GP1 or N-β2GP1 (0.1 mg/mL) was added to 96 wellplates coated with Matrigel. Images were recorded after 4 and 24 hoursincubation at 37° C. N-β2GP1 caused cells to wander without direction onthe plate, while control cultures incubated in medium alone or in mediasupplemented with intact β2GP1 began to assemble into capillary likestructures within ˜4 hours. Differences in the architecture of the fullyformed tubes were also evident after 24 hours.

FIG. 7. Ability of β2GP1 to inhibit endothelial cell migration. Toexamine migration, BFGF chemo-attractant was added to the lower chambersof the transwells of a 24-well polycarbonate Boyden chamber. Next, HUVECwere resuspended with or without β2GP1 or N-β2GP1 (100 μg/mL or 200μg/mL), and added to upper chamber. After a 5 hour incubation at 37° C.,the transwells were fixed and stained and the number of cells thatmigrated was counted. N-β2GP1 at both concentrations was able to inhibitmigration of endothelial cells in the Boyden chamber invasion assay,while intact β2GP1 was without effect.

FIG. 8. Growth factor and chemically-induced blood vessel dilation(angioectasia). Normal mice were anesthetized and the mesentery wasdraped on the specimen stage of a dissecting microscope. After wettingthe area of interest with vascular endothelial cell growth factor (VEGF)or nitroglycerin (NG), images were collected in real-time for 1 hour.VEGF: Areas of obvious dilation are marked with arrows. Note theincreased tortuousness of vessels after the addition of VEGF, marked bythe squiggly trace following some vessels in the VEGF frame (lowerleft), which was copied and superimposed on the same vessel in the zerotime image (upper left). NG: Obvious areas of dilated vessels are alsomarked, although the magnitude of change is not as great as thatobtained with VEGF. The circled area marks an area were small vesselsbecame visible after the addition of NG.

FIG. 9. Kinetics of VEGF-induced angioectasia. Control mice wereinjected intravenously with fluorescein-conjugated human serum albumin(FITC-HAS), anesthetized, and prepared as described in FIG. 8. VEGF wasapplied at time zero, the area was videotaped and frames of interestwere digitized and captured. There are several notable features in thissequence. First, note the relatively sparseness of the fluorescentvessels in the circled area before the addition of VEGF. At 1 minute,the density of fluorescent vessels in this area increased significantlyand then began to gradually disappear over the next 5 minutes. Second,note the fluorescent blood vessel marked with the white arrow before theaddition of VEGF (−1 min). Within 2 minutes after the addition of VEGFthe fluorescence disappeared (due to the vessel filling with red bloodcells and quenching of FITC-HSA). At 7 minutes, a diffuse area offluorescence seems to appear which may represent increased vesselpermeability and leakage of small plasma proteins (FITC-HSA) to thesurrounding tissue. The light image taken at 7 minutes shows a newlyvisible (red blood cell filled) blood vessel.

FIG. 10. Mice treated with intact β2GP1 and N-β2GP1 were injected withFITC-HSA. The mice were then prepared as described in. FIG. 8 andvideotaped throughout the application of VEGF. The arrows on the leftpoint to the same vessel (orientation problems after moving the animals)1 hour after adding VEGF. Note absence of dilation in the mice treatedwith N-β2GP1. These results suggest that N-β2GP1 inhibits growthfactor-Induced angioectasia in vivo.

FIG. 11. Vessel pattern in VEGF-treated mesentery. To better describetypical patterns of fluorescent and “red” vessels, multiple frames fromVEGF-induced angioectasia in mice injected with intact β2GP1, N-β2GP1,and HSA were carefully traced. Mice injected with intact β2GP1 or HSAgenerally showed a small number of fluorescent vessels that immediatelyincreased upon addition of VEGF. No fluorescent vessels were seen inmice pretreated with N-β2GP1. Top, before; middle, 1 minute after; andbottom, 7 minutes after the addition of VEGF, respectively.

FIG. 12. Mice treated with intact β2GP1, N-β2GP1, and HSA were preparedas described in. FIG. 8 and videotaped before and after (0.5 hours) theapplication of nitroglycerin. The circles mark the areas of obviousvessel dilation. These results suggest that β2GP1 does not inhibitchemically-induced angioectasia in vivo.

FIG. 13. Construction of β2GP1 domain deleted mutants. Deletionconstructs were initiated from the linker region between thedisulfide-linked domains in order to keep distal domains intact. Theapproximate locations of cut sites are illustrated by bold vertical barsin panel A. Panel B shows the schematic diagram of the primer start siteof the respective PCR amplification products. Panel C maps the domainspresent in the different constructs with their respective N-terminalamino acid sequence. Constructs were based on human liver cDNA EMBLaccession no. X57847.

FIG. 14. Identification of PCR products set forth in FIG. 12. The PCRamplified products representing the sequential domains described inExample 4 were analyzed by agarose gel electrophoresis (cDNA fragments979 bp, 790 bp, 625 bp, 430 bp, and 253 by in length, respectively). Thefar left and right lanes are 1 kb and 100 by ladders, respectively.

FIG. 15. Analysis of recombinant proteins of β2GP1 domain deletedmutants. Gels 1 and 4 show secreted recombinant full length protein anddomain V, respectively. Gels 2 and 3 show constitutively-expressedrecombinant domains 3-5 and 4-5, respectively (see FIG. 12). Asterisksindicate the recombinant products.

FIG. 16. Generation of β2GP1 domain-deleted mutants with trypsin. Twomajor fragments were generated with lipid-bound β2GP1 digested withtrypsin: a ˜30 kDa fragment (*I) with the N-terminal sequenceDTAVFECLPQH, and a ˜40 kDa (*2) fragment with the N-terminal sequenceYTTFEYPNTIS. These two peptide fragments are consistent with singlepolypeptides encompassing domains III-V and II-V, respectively. Twomajor fragments were also generated with native unbound β2GP1: a ˜10 kDafragment (*3) and a ˜40 kDa fragment (*4), both of which had theN-terminal sequence GRTCPKPDDLP. This sequence is consistent withpolypeptides encompassing domain I and domains I-IV. Molecular weightmarkers (arrows) are 121, 86, 69, 52, 40, 28, 22, 17 and 9 kDa.

FIG. 17. Hypothetical β2GP1-dependent mechanism for the inhibition ofendothelial cell angiogenesis/angioectasia. (A) β2GP1 in tumor vascularendothelial cells binds to both annexin II and PS, and preventsPS/annexin II interactions. (B) Cleavage of β2GP1 with endogenousplasmin results in release of PS. Alternatively, exogenously-suppliedN-β2GP1 competes for endogenous (intact) β2GP1 binding to annexin II.This results in its dissociation from PS because of its low affinitybinding (˜10⁻⁶ M) in the absence of multivalent interactions. C) Thisenables the association of PS with annexin II that leads to a sequenceof intracellular events that culminates in inhibition of angiogenesis.

FIG. 18. Left panel. Vascular endothelial cells were incubated withintact β2GP1 (top) or N-β2GP1 (bottom), washed, and then stained withfluorescein-conjugated anti-β2GP1 (Left, fluorescence; right: light).Both intact β2GP1 and N-β2GP1 bind endothelial cells. Middle panel.ELISA plates coated with intact β2GP1 or N-β2GP1 were blocked with 1%ovalbumin and biotin labeled annexin was added. The plates were washedafter 1 hr and developed with peroxidase-avidin. Both intact β2GP1 andN-β2GP1 bind annexin. Right panel. Phosphatidylserine-coated ELISAplates were blocked with 1% ovalbumin and incubated with intact β2GP1 orN-β2GP1. Protein binding to the plates was determined with rabbit β2GP1antibodies followed by peroxidase-conjugated anti-rabbit lg. Note thatonly intact β2GP1 was able to bind lipid, and the comparativelysignificant decrease in the binding of N-β2GP1.

FIG. 19. Binding of β2GP1 to endothelial cells. Both intact β2GP1 andN-β2GP1 were labeled with ¹²⁵I, and added in the presence of increasingconcentrations of unlabeled N-β2GP1 to endothelial cells. Uptake wasdetermined by scintillation counting. Increasing concentrations ofunlabeled N-β2GP1 competed for the binding of both ¹²⁵I-labeledproteins, suggesting that both intact β2GP1 and N-β2GP1 bind to the sameendothelial cell binding site.

FIG. 20. Potential mechanism by which N-β2GP1 inhibits endothelial cellangiogenesis/angioectasia. The possibilities are: (i) direct binding toVEGF, which inhibits VEGF binding to its receptor; (ii) binding toVEGF-R (receptor antagonist); or (iii) binding to Tie-1 (receptoragonist).

DETAILED DESCRIPTION OF THE INVENTION

Beta-2-glycoprotein 1 (β2GP1) is a protein that has antiangiogenicproperties. The present disclosure relates to the inhibition, treatment,or prevention of unwanted endothelial cell proliferation, cordformation, and cell migration, as well as angiogenesis and angioectasia.Angiogenic diseases that may be treated by using β2GP1 include but arenot limited to, diabetic retinopathy, retrolental fibroplasia, trachoma,neovascular glaucoma, psoriases, angio-fibromas, immune and non-immuneinflammation, capillary formation within atherosclerotic plaques,hemangiomas, excessive wound repair, solid tumors, Kaposi's sarcoma, andthe like. In a preferred embodiment, the present disclosure relates tothe use of β2GP1 to inhibit angiogenesis and angioectasia at the site oftumorigenesis or tumor growth, which can then prevent or inhibit tumorgrowth.

β2GP1 includes both the intact form of β2GP1 and N-1:32 GP1. One form ofβ2GP1 polypeptide (a human amino acid sequence) is set forth below:

SEQ ID: 1 GRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGGMRKFICPLTGLWPINTLKCTPRVCPFAGILENGAVRYTTFEYPNTISFSCNTGFYLNGADSAKCTEEGKWSPELPVCAPIICPPPSIPTFATLRVYKPSAGNNSLYRDTAVFECLPQHAMFGNDTITCTTHGNWTKLPECREVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSLDGPEEIECTKLGNWSAMPSCKASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFCKNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWKTDASDVKPC

However, the β2GP1 polypeptide is not limited to the use of the aboveexemplary sequence. Indeed, many other β2GP1 sequences are known in theart, and genetic sequences can vary between different species andindividuals. For example, nucleic acid, cDNA, and protein sequences havebeen determined for human, rat, bovine, and mouse β2GP1, and reveal ahigh degree of homology in the β2GP1 sequences between species (seee.g., Steinkasserer et al., Biochem. J. 277:387-391, 1991; Lozier etal., Proc. Natl. Acad. Sci. (USA) 81:3640-3645, 1984; Kristensen et al.,FEBS Lett. 289(2):183-186, 1991; Matsuura et al., Int. Immunol.3(12):1217-1221, 1991; Mehdi et al., Gene 108:293-298, 1991; Day et al.,Int. J. Clin. Lab. Res. 21(3):256-263, 1992; Aoyama et al., NucleicAcids Res. 17:6401, 1989; Nonaka et al., Genomics 13:1082-1087, 1992;incorporated herein by reference). This natural scope of allelicvariation is included within the scope of the present disclosure. It isto be understood that the spirit of the present disclosure iscontemplated to include any derivatives of β2GP1 that inhibit, treat, orprevent unwanted endothelial cell proliferation and/or angiogenesis. Theβ2GP1 polypeptide can also include other domains, such as epitope tagsand His tags (e.g., the protein can be a fusion protein). The presentdisclosure also includes genes and cDNAs that code for β2GP1 andproteins that are expressed by those genes.

Within the context of the present disclosure, the β2GP1 polypeptide maybe or comprise insertion, deletion, or substitution mutants of a knownβ2GP1 sequence or derivative thereof. Included as analogs, derivatives,and fragments of β2GP1 are polypeptides with conservative amino acidsubstitutions or non-conservative amino-acid substitutions, deletions,or insertions, which do not significantly reduce the anti-angiogenicactivity of the β2GP1. Preferably, any substitution is conservative inthat it minimally disrupts the biochemical properties and/orbiologically functional properties of the β2GP1 polypeptide. Thus, wheremutations are introduced to substitute amino acid residues,positively-charged residues (H, K, and R) are preferably substitutedwith positively-charged residues; negatively-charged residues (D and E)are preferably substituted with negatively-charged residues; neutralpolar residues (C, G, N, Q, S, T, and Y) are preferably substituted withneutral polar residues; and neutral non-polar residues (A, F, I, L, M,P, V, and W) are preferably substituted with neutral non-polar residues.The present disclosure also includes β2GP1 attached to a carrier orligand, or coupled to carbohydrates such as PEG or protein carriers aslong as the β2GP1 retains anti-angiogenic activity.

In the methods of treatment disclosed herein, the administration ofβ2GP1 may be for either “prophylactic” or “therapeutic” purposes. Whenprovided prophylactically, β2GP1 is provided in advance of any symptom.The prophylactic administration of β2GP1 serves to prevent or inhibitunwanted endothelial cell proliferation and/or angiogenesis at a site.When provided therapeutically, β2GP1 is provided at (or after) the onsetof one or more symptoms or indications of unwanted endothelial cellproliferation and/or angiogenesis. Thus, β2GP1 may be provided eitherprior to the anticipated angiogenesis at a site or after angiogenesishas begun at a site.

Without being bound by any particular theory, β2GP1 may inhibitangiogenesis, in part, by attenuating the migration of endothelialcells, thus reducing or preventing neovascularization into a tissue.Thus, the disclosure provides a method of inhibiting endothelial cellmigration by providing β2GP1 to such cells. Aside from attenuatingangiogenesis, β2GP1 is useful for treating disorders associated withstimulation of endothelial cell migration such as intestinal adhesions,Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars(e.g., keloids). β2GP1 is also useful for treating pathologicalangiogenesis, which is characterized by the persistent proliferation ofendothelial cells, and is a prominent feature of diseases such as, butnot limited to, rheumatoid arthritis, scleroderma, lupus erythematosus,and psoriasis. Angiogenesis associated with wound repair may also betreated using β2GP1. Although controlled angiogenesis occurs during avariety of physiological processes, such as embryogenesis and woundrepair, wound healing can be associated with excessiveneovascularization and may result in keloid formation, excessive dermalscarring at sites of skin trauma or surgical sites, and othercomplications. Thus, it may appropriate in a number of circumstances toinhibit physiological neovascularization to prevent or alleviatecomplications of excessive neovascularization.

The present disclosure is useful in inhibiting angiogenic function oftarget cells, tissues, or organisms. In particular embodiments, at leastone β2GP1 may be transfected into at least one cell, tissue, ororganism. In particular aspects, the β2GP1 is transcribed, and in morespecific aspects, translated into a protein, polypeptide or peptide inat least one cell, tissue, or organism. As used herein, the terms“cell,” “cell line,” and “cell culture” may be used interchangeably. Allof these term also include their progeny, which is any and allsubsequent generations. It is understood that all progeny may not beidentical due to deliberate or inadvertent mutations. In the context ofexpressing a heterologous nucleic acid sequence, “host cell” refers to aprokaryotic or eukaryotic cell, and it includes any transformableorganisms that is capable of replicating a vector or construct and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

In the present disclosure, target cells may be endothelial cells, bothin vitro and in vivo. β2GP1 can be provided to endothelial cellsassociated with the tissue of interest. As used herein, a tissue ofinterest includes, but is not limited to dermis, epidermis, endometrium,retina, surgical wound, gastrointestinal tract, umbilical cord, liver,kidney, reproductive system, lymphoid system, central nervous system,breast tissue, urinary tract, circulatory system, bone, muscle,respiratory tract, skin, endocrine system, brain, neoplasm, or tumortissue. The endothelial cells can be cells in the tissue of interest,exogenous cells introduced into the tissue, or neighboring cells notwithin the tissue. Thus, for example, the cells can be cells of thetissue, with β2GP1 provided to them such that the β2GP1 contacts thecells. Alternatively, the cells can be introduced into the tissue, inwhich case the β2GP1 or an expression vector or construct of β2GP1 canbe transferred to the cells before they are so introduced into thetissue (e.g., in vitro), as well as being transferred in situ afterintroduction into the tissue.

The cell or cells to be transformed with a β2GP1 vector or construct maybe comprised in a tissue. The target tissue may be part or separatedfrom an organism. In certain embodiments, a tissue may comprise, but isnot limited to, skin, bone, neuron, axon, cartilage, blood vessel,cornea, muscle, fascia, brain, prostate, breast, endometrium, lung,pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon,skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney,peripheral blood, embryonic, ascite tissue, and all cancers thereof.Tissue with which endothelial cells are associated are also contemplatedherein, and β2GP1 can be used in any tissue in which it is desired toinhibit the migration or expansion of endothelia (e.g., for inhibitingangiogenesis).

In certain embodiments, the cell or tissue may be comprised in at leastone organism. In certain embodiments, the organism may be, but is notlimited to, an eukaryote, an animal, a vertebrate, a primate (e.g.,monkey, lemur, gorilla, chimp, human), a canine, a feline, a bovine, anequine, an ovine, a murine, a caprine, a porcine species, and the like.

A. Treatment of Angiogenic Diseases with β2GP1

A variety of tissues may be treated with β2GP1 to inhibit theproliferation, cord formation, cell migration, or differentiation ofendothelial cells and/or angiogenesis. In a preferred embodiment, thetissue is a neoplasm (e.g., a cancerous tumor), in which β2GP1 inhibitsthe growth of blood vessels within and to the neoplasm. In anotherpreferred embodiment, β2GP1 inhibits metastasis of the neoplasm. As usedherein, the term “neoplasm” refers to any type of malignant or benignneoplasm, including any type of diffuse neoplasm such as leukemia, aswell as malignant or benign cancers and tumors (including any carcinoma,sarcoma, or adenoma). A neoplasm is abnormal tissue that grows bycellular proliferation more rapidly than normal, and can continue togrow after the stimuli that initiated the new growth has ceased. Aneoplasm may also have partial or complete lack of structuralorganization and functional coordination with normal tissue.Specifically contemplated neoplasms are, for example, tumors such astumors of the mammary, pituitary, thyroid, prostate gland, brain, liver,meninges, bone, ovary, uterus, cervix, and the like.

Additional specifically contemplated neoplasms include, but are notlimited to, adenocarcinoma, adenoma, astrocytoma, bladder tumor, bonecarcinoma, brain carcinoma, Burkitt lymphoma, Kaposi Sarcoma,non-Hodgkins lymphoma, Hodgkins lymphoma, gastric tumor, breastcarcinoma, cervical carcinoma, colon carcinoma, kidney carcinoma, livercarcinoma, lung carcinoma, ovarian carcinoma, pancreatic carcinoma,prostate carcinoma, rectal carcinoma, skin carcinoma, stomach carcinoma,testis carcinoma, thyroid carcinoma, chondrosarcoma, choriocarcinoma,fibroma, fibrosarcoma, glioblastoma, glioma, hepatoma, histiocytoma,leiomyoblastoma, leiomyosarcoma, leukemia, lymphoma, liposarcoma cell,mammary carcinoma, medulloblastoma, melanoma, metastases, muscle tumor,myeloma, ovarian carcinoma, plasmacytoma, neuroblastoma, neuroglioma,osteogenic sarcoma, pancreatic tumor, pituitary carcinoma, renal tumors,retinoblastoma, rhabdomyosarcoma, sarcoma, testicular tumor, thymoma,uterine carcinoma, Wilms' tumor, and the like.

Inhibiting the growth of blood vessels within a neoplasm will preventsufficient nutrients and oxygen from being supplied to the neoplasm tosupport growth beyond a given size. Thus, β2GP1 can prevent thenucleation of neoplasms from cancerous cells already present due togenetic predisposition (e.g., BRCA-1 mutation carriers, Li Fraumenipatients with p53 mutations, etc.) or the presence of externalcarcinogens (e.g., tobacco, alcohol, industrial solvents, etc.). Asidefrom preventing neoplastic disease or tumerogenesis, β2GP1 can alsoretard the growth of existing neoplasms, thus rendering them more easilycontained and excised. This application is highly advantageous fortreating neoplasms that are difficult to operate on (e.g., brain orprostate tumors). Moreover, minimizing the number of blood vesselswithin an existing neoplasm lessens the probability that the neoplasmwill metastasize.

In treating neoplasms, β2GP1 can be used alone or in conjunction withother therapeutic agents and/or treatments to control the growth of aneoplasm. Such therapies are particularly useful when the subject to betreated has a large preexisting tumor mass which is well vascularized.Indeed, employing β2GP1 can potentiate the response of some neoplasms toother therapeutic agents and/or treatments therapies. For example, theadministration of β2GP1 optionally can be employed as a pretreatment for(e.g., for about a week in advance of), and/or continued during, achemotherapeutic, anti-neoplastic, or radiation regimen. Additionaltherapeutic agents specifically contemplated by the present disclosureinclude but not limited to anti-neoplastic agents, chemotherapeuticagents, or even cocktails. Additional neoplastic therapies specificallycontemplated by the present disclosure include but are not limited tosurgery, chemotherapy, radiation therapy, pharmacotherapy, gene therapy,and immunotherapy treatments. Radiation therapy includes but is notlimited to ionizing radiation; gamma radiation from radioactive isotopessuch as cobalt-60, radium, radon, iridium, or electrically generatedroentgen rays; radiation by external beam, implant, pellet, or seed; orvariants thereof. β2GP1 may be administered to a patient before, during,and/or after other available neoplastic therapies. Additionally,separate administration of β2GP1 from the other therapeutic agentsand/or treatments, or even an administration which is spaced in time, iscontemplated by the present disclosure.

The present disclosure further encompasses methods for treating aneoplasm by administering to the subject a pharmaceutical compositionthat includes β2GP1 and one or more additional therapeutic agents,including but not limited to anti-neoplastic agents, chemotherapeuticagents, or even cocktails, to treat the neoplasm. Such a pharmaceuticalcomposition may be used to inhibit, prevent, or suppress the growth of aneoplasm. The therapeutic agents used in combination with β2GP1 to treata neoplasm can be presented to the subject in a separate formulation oras a mixture. Thus, separate administration of a therapeutic agent oreven an administration which is spaced in time is contemplated by thepresent disclosure, particularly when the therapeutic agent and β2GP1have a synergistic therapeutic action.

Examples of therapeutic agents useful in the treatment of neoplasmsinclude, but are not limited to, taxol, tamoxifen, taxotere,doxorubicin, cisplatin, cyclophosphamide, gancyclovir, paclitaxel,methotrexate, mechlorethamine, aldesleukin, altretamine, amsacrine,azacitidine, ifosfamide, melphalan, chlorambucil, hexamethylmelamine,thiotepa, triethylenethiophosphoramide, busulfan, carmustine, lomustine,semustine, streptozocin, dacarbazine, fluorouacil, floxuridine,fludarabine, goserelin, cytarabine, levamisole, mercaptopurine,thioguanine, pentostatin, pipobroman, vinblastine, vincristine,vindesine, etoposide, teniposide, dactinomycin, daunorubicin,estramustine, filgrastim, bleomycin, plicamycin, uracil mustard,mitomycin, L-asparaginase, interferon-alpha, carboplatin, mitoxantrone,hydroxyurea, procarbazine, mitotane, aminoglutethimide, prednisone,hydroxyprogesterone caproate, medroxyprogesterone acetate, megastrolacetate, diethylstilbestrol, ethinyl estradiol, testosterone propionate,fluoxymesterone, flutamide, and leuprolide, an interferon, a tumornecrosis factor, a radiation implant such as a pellet or seed, orvariants thereof.

In treating neoplasms, β2GP1 can also be used alone or in conjunctionwith other antiangiogenic agents to control the growth of a neoplasm.Indeed, employing β2GP1 and one or more additional antiangiogenic agentsmay potentiate the response of some neoplasms to these agents.Additional antiangiogenic agents specifically contemplated by thepresent disclosure include but not limited to angiostatin, endostatin,trastuzumab, TGF-β, thrombospondin, IL-1, IFN-γ, IFN-α, tissue inhibitorof metalloproteinase-1 (TIMP-1), platelet factor 4 (PF4), protamine,retinoic acid, AGM-1470, fumagillin, tyrosine kinase inhibitor, aninhibitor of epidermal-derived growth factor, an inhibitor offibroblast-derived growth factor, an inhibitor of platelet derivedgrowth factor, an MMP (matrix metalloprotease) inhibitor, an integrinblocker, interleukin-12, pentosan polysulfate, a cyclooxygenaseinhibitor, fibronectin, laminin, prolactin, carboxyamidotriazole,combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol,thalidomide, troponin-1, and an antibody to VEGF

In addition, β2GP1 can be used in conjunction with antibodies andpolypeptides that block integrin engagement, proteins and smallmolecules that inhibit metalloproteinases (e.g., marmistat), agents thatblock phosphorylation cascades within endothelial cells (e.g.,herbamycin), dominant negative receptors for known inducers ofangiogenesis, anti-inflammatory agents (e.g., ibuprofen, aspirin,prednisone), antibodies against inducers of angiogenesis or othercompounds that block their activity (e.g., suramin), or other compounds(e.g., retinoids, IL-4, interferons, etc.) acting by other means.Indeed, because other antiangiogenic factors may inhibit angiogenesis bydifferent mechanisms, employing β2GP1 in combination with otherantiangiogenic agents may potentiate a more potent (and potentiallysynergistic) inhibition of angiogenesis within the desired tissue. β2GP1may be administered to a patient before, during, and/or after theadministration of other available antiangiogenic agents to a subject.Additionally, separate administration of β2GP1 from the otherantiangiogenic agents, or even an administration which is spaced intime, is contemplated by the present disclosure.

The present disclosure further encompasses methods for treating aneoplasm by administering to the subject a pharmaceutical compositionthat includes β2GP1 and one or more additional antiangiogenic agents totreat the neoplasm. Such a pharmaceutical composition may be used toinhibit, prevent, or suppress the growth of a neoplasm. Theantiangiogenic agents used in combination with β2GP1 to treat a neoplasmcan be presented to the subject in a separate formulation or as amixture. Thus, separate administration of a antiangiogenic agent or evenan administration which is spaced in time is contemplated by the presentdisclosure, particularly when the antiangiogenic agent and β2GP1 have asynergistic therapeutic action.

Where β2GP1 is applied to other tissues, the prevention ofneovascularization effectively treats a host of disorders. Thus, forexample, β2GP1 can be used as part of a treatment for disorders of bloodvessels (e.g., hemangiomas and capillary proliferation withinatherosclerotic plaques), muscle diseases (e.g., myocardial angiogenesisor angiogenesis within smooth muscles), joints (e.g., arthritis,hemophiliac joints, etc.), and other disorders associated withangiogenesis (e.g., Osler-Webber Syndrome, plaque neovascularization,telangiectasia, angiofibroma, wound granularization, etc.).

In one embodiment, the tissue can be eye tissue, in which case thepresence of β2GP1 will inhibit novel angiogenesis associated with avariety of disorders of the eye. For example, β2GP1 is useful fortreating eye injury, hypoxia, infection, surgery, laser surgery,diabetes, retinoblastoma, or other diseases or disorders of the eye. Inthis respect, the method is useful for preventing blindness or retardingloss of vision associated with a variety of eye diseases.

In another embodiment, the tissue is skin tissue, in which case thepresence of β2GP1 prevents neovascularization associated with severalskin diseases. For example, the inventive method is useful for treatingdiseases and disorders such as psoriasis, scleroderma, tumors of theskin, neovascularization as a consequence of infection (e.g., catscratch disease, bacterial ulceration, etc.), or other skin disorders.Where β2GP1 is provided to the skin, it can be provided to the surfaceof the skin or to skin tissue beneath the skin's surface. Furthermore,transfer of β2GP1 to skin of a mammal may also stimulate the growth ofhair in the skin.

The present disclosure also provides a method for determining theprognosis of a tumor by assaying for the presence of β2GP1 within thetumor. The method involves obtaining tissue or fluid from the tumor anddetecting the presence or absence of β2GP1 within the tissue or fluid.Greater β2GP1 concentration within the tumor may correlate with a lesserlikelihood that the tumor is undergoing angiogenesis, depending on thetumor assayed. The method can employ an assay for the presence of β2GP1gene expression (e.g., via rtPCR, Northern hybridization, in situhybridization, etc.). Alternatively, the method can employ an assay forthe presence of β2GP1 polypeptides (e.g., immunological assays, β2GP1purification and PAGE analysis, etc.).

B. β2GP1 Proteins, Polypeptides, and Peptides

As discussed herein, β2GP1 is a polypeptide. Contemplated by the presentdisclosure are methods for providing β2GP1 by supplying a β2GP1polypeptide to cells, tissues, or organisms of interest (e.g., within asuitable composition). Any suitable method known to those of skill inthe art can be employed to obtain a β2GP1 polypeptide for use in thepresent disclosure, and β2GP1 may be obtained from natural, recombinant,or synthetic sources. For example, β2GP1 can be purified from animalplasma, or β2GP1 can be produced recombinantly, chemically, orenzymatically.

A particularly good source of naturally occurring β2GP1 is human plasma.Plasma can be obtained from regional blood centers without regard torace, gender, or ethnic background. Examples of protocols for purifyingβ2GP1 from human plasma known to those of skill in the art include butare not limited to perchloric acid precipitation, ion-exchange, andheparin affinity chromatogaphy (Wurm, H., Int. J. Biochem. 16:511-515,1984; Polz et al., Int. J. Biochem. 11:265-270, 1980; Schousboe, I.,Biochim. Biophys. Acta 579:396-408, 1979; incorporated herein byreference). N-β2GP1 (Lys-317/Thr-318) may also be prepared usingtechniques known to those of skill in the art. For example, N-β2GP1 canbe isolated from human plasma (see Horbach et al., Throm. Haemostasis81:87-95, 1999, incorporated herein by reference), or N-β2GP1 may beprepared from plasmin and purified by an additional heparin affinitychromatography step (Horbach et al., Throm. Haemostasis 81:87-95, 1999;Ohkura et al., Blood 91:4173-4179, 1998; incorporated herein byreference). Additionally, N-β2GP1 can also be prepared byproteolytically cleaving intact β2GP1 at Lys-317 and Thr-318 in vitrowith the enzymes factor Xa or elastase. Unwanted proteolytic cleavage ofintact β2GP1 at Lys-317 and Thr-318 can be prevented by, for example,pretreating plasma with perchloric acid precipitation. Other protocolsfor purifying β2GP1 polypeptides are known in the art.

Yet another method of producing β2GP1, or biologically active fragmentsthereof, is by peptide synthesis. A β2GP1 polypeptide is identified viaSDS-PAGE as a protein of about 50 kDa. β2GP1 polypeptides can besynthesized using standard direct peptide synthesizing techniques knownto those of skill in the art, such as via solid-phase synthesis (see,e.g., Barany et al., Int. J. Peptide Protein Res., 30:705-739, 1987;U.S. Pat. No. 5,424,398; Solid Phase Peptide Synthesis: A PracticalApproach E. Atherton and R. C. Sheppard, IRL Press, Oxford England;incorporated herein by reference). Similarly, multiple fragments can besynthesized which are subsequently linked together to form largerfragments.

The present disclosure provides for purified, and in preferredembodiments, substantially purified, β2GP1 polypeptides. As used hereinthe term “β2GP1 polypeptide” includes β2GP1 proteins, polypeptides, orpeptides. The term “purified β2GP1 polypeptides” as used herein isintended to refer to a β2GP1 proteinaceous composition, isolatable fromnatural, recombinant, or synthetic sources, wherein the β2GP1polypeptide is purified to any degree relative to itsnaturally-obtainable state, i.e., relative to its purity within acellular extract. As used herein, the term “proteinaceous composition”encompasses the terms protein, polypeptide, and peptide. A β2GP1polypeptide therefore also refers to a wild-type or mutant β2GP1polypeptide free from the environment in which it naturally occurs. Asused herein “wild-type” refers to the naturally occurring sequence of anucleic acid at a genetic locus in the genome of an organism, or asequence transcribed or translated from such a nucleic acid. The term“wild-type” also may refer to an amino acid sequence encoded by anucleic acid. Since a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring allele(s). The β2GP1 polypeptides may befull length proteins, for example 326 amino acids in length, or they mayalso be less then full length proteins, such as individual domains,regions, or even epitopic peptides. The most preferred less than fulllength β2GP1 proteins are those containing predicted immunogenic sitesand those containing the functional domains identified herein.

Generally, “purified” will refer to a β2GP1 polypeptide composition thathas been subjected to fractionation to remove various non-β2GP1polypeptides, and which composition substantially retains its β2GP1antiangiogenic activity. The term “substantially purified” refers to acomposition in which the β2GP1 polypeptide forms the major component ofthe composition, such as constituting about 50% of the proteins in thecomposition or more. In preferred embodiments, a substantially purifiedprotein will constitute more than 60%, 70%, 80%, 90%, 95%, 99%, or evenmore of the proteins in the composition. A polypeptide that is “purifiedto homogeneity,” as applied to the present disclosure, means that thepolypeptide has a level of purity where the polypeptide is substantiallyfree from other proteins and biological components. For example, apurified polypeptide will often be sufficiently free of other proteincomponents so that degradative sequencing may be performed successfully.

Various methods for quantifying the degree of purification of β2GP1polypeptides will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificβ2GP1 protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis. Assessing thenumber of polypeptides within a fraction by SDS/PAGE analysis will oftenbe preferred in the context of the present disclosure because it is astraightforward protocol. The activity of a fraction may also bedetermined by a number of methods known to those of skill in the art,including but not limited to affinity chromatography, gelatinenzymography, or zymography.

To purify a β2GP1 polypeptide, a natural or recombinant compositioncomprising at least some β2GP1 polypeptide will be subjected tofractionation to remove various non-β2GP1 components from thecomposition. Techniques suitable for use in protein purification arewell known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite, lectinaffinity and other affinity chromatography steps; isoelectric focusing;gel electrophoresis; and combinations of such and other techniques.Additionally, recombinant β2GP1 may be purified from cell-free lysatesby affinity chromatography on anti-β2GP1-sepharose. Another example isthe purification of β2GP1 fusion proteins using specific bindingpartners. Such purification methods are routine in the art. Since DNAcoding sequences for β2GP1 are known, any fusion protein purificationmethod can be practiced.

Isolated β2GP1 polypeptides can be conclusively identified by N-terminalamino acid sequencing (intact β2GP1: single N-terminal sequence (GRTCPK. . . ); N-β2GP1: two N-terminal sequences (GRCPTK . . . and TDASD . . .)). β2GP1 polypeptides can also be labeled using a variety of methodsknown to those of skill in the art. For example, polypeptides can belabeled by iodination with ¹²⁵I using iodogen or iodobeads as previouslydescribed (Ma et al., J. Biol. Chem. 275:15541-15548, 2000; incorporatedherein by reference) or by direct coupling to appropriately activated(N-hydroxysuccinimide-, maleimide- or hydrazine-) fluorophores. β2GP1binds endothelial cells through annexin II, which can be purified asdescribed in Khanna et al., Biochemistry 29:4852-4862, 1990,incorporated herein by reference.

C. Recombinant Vectors, Host Cells and Expression

Recombinant vectors form important further aspects of the presentdisclosure. In methods disclosed herein, β2GP1 polypeptide can beprovided to a cell, tissue, or organism of interest by transferring anexpression vector or construct that includes a nucleic acid encodingβ2GP1 to cells associated with the tissue or organism of interest. Theterm “expression vector or construct” refers to any type of geneticconstruct containing a nucleic acid coding for a gene product in whichpart or all of the nucleic acid encoding sequence is capable of beingtranscribed. The transcript may be translated into a protein, but itneed not be. Thus, in certain embodiments, expression includes bothtranscription of a gene and translation of a RNA into a gene product. Anexpression vector or construct may include any desired DNA sequence thatcan be incorporated into the genome of a cell, including but not limitedto genes or DNA sequences that are not normally present in the genome,genes or DNA sequences that are normally present, but are not normallytranscribed and translated (“expressed”) in a given genome, or any othergenes or DNA sequences that one desires to introduce into the genome.This may include genes or DNA sequences that are normally present in thegenome of the cell, tissue, or organism of interest, but which onedesires to have altered in expression, or which one desires to introducein an altered or variant form.

Methods for producing β2GP1 polypeptide recombinantly are well to thoseof skill in the art. One example of a method for producing β2GP1 usingrecombinant DNA techniques entails the steps of (1) inserting the β2GP1gene or cDNA into an appropriate vector such as an expression vector,(2) inserting the gene-containing vector into a microorganism or otherexpression system capable of expressing the β2GP1 gene, and (3)isolating the recombinantly produced β2GP1. The above techniques aremore fully described in laboratory manuals such as Sambrook et al.(1989), supra, incorporated herein by reference. Examples of methods forgenerating recombinant β2GP1 are disclosed in Kouts et al., FEBS Lett.326:105-108, 1993; Chen et al., Chinese Med. J. 112:67-71, 1999; andKristensen et al., FEBS Lett. 289:183-186, 1991, incorporated herein byreference.

Any suitable vector or construct can be employed as a recombinantvector, many of which are known in the art. Examples of such vectorsinclude but are not limited to naked DNA vectors (such asoligonucleotides or plasmids), viral vectors such as adeno-associatedviral vectors (Bern and Giraud, Ann. N.Y. Acad. Sci. 772:95-104, 1995;incorporated herein by reference), adenoviral vectors (Bain et al., GeneTherapy Suppl. 1; S68, 1994; incorporated herein by reference),herpesvirus vectors (Fink et al., Ann. Rev. Neurosci. 19:265-87, 1996;incorporated herein by reference), packaged amplicons (Carew et al.,Mol. Ther. 4:250-256, 2001; incorporated herein by reference), pappilomavirus vectors, picornavirus vectors, polyoma virus vectors, retroviralvectors, SV40 viral vectors, vaccinia virus vectors, and the like.

In one embodiment, the expression vector or construct will be introducedinto the cell, tissue, or organism of interest to produce and secretethe β2GP1 polypeptide, for example, to endothelial cells within thetissue, such that angiogenesis is inhibited or attenuated within thetissue or organism of interest. Coding sequences for β2GP1 genes, cDNAs,and polypeptides are known, and others can be deduced from the geneticsequences discussed herein. Thus, β2GP1 expression vectors or constructswill typically employ coding sequences homologous to these knownsequences, e.g., they will hybridize to at least a fragment of the knownsequences under at least mild stringency conditions, more preferablyunder moderate stringency conditions, most preferably under highstringency conditions (employing the definitions of mild, moderate, andhigh stringency as set forth in Sambrook et al., (1989) MolecularCloning: A Laboratory Manual, 2d ed., 1989, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., incorporated herein byreference).

In addition to the β2GP1 coding sequence, an expression vector orconstruct may include regulatory elements that drive expression of β2GP1by providing transcriptional and translational initiation regionsassociated with gene expression in the cell or tissue of interest, andfunctional transcriptional and translational termination regions.Particularly useful vectors or constructs are contemplated to be thosein which the coding portion of the DNA segment, whether encoding a fulllength protein, polypeptide, or smaller peptide, is positioned under thetranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the transcriptional machinery of the cell, orintroduced transcriptional machinery, required to initiate the specifictranscription of a gene. The phrases “operatively positioned,” “undercontrol,” or “under transcriptional control” means that the promoter isin the correct location and orientation in relation to the nucleic acidto control RNA polymerase initiation and expression of the gene. As usedherein, the term “operable linkage” refers to a linkage ofpolynucleotide elements in a functional relationship.

For example, a promoter or enhancer is in operable linkage to a codingsequence if it affects and/or regulates the transcription of the codingsequence. As long as this operable linkage is maintained, the expressioncassette can include more than one gene, such as multiple genesseparated by ribosome entry sites. The promoter may be in the form ofthe promoter that is naturally associated with a β2GP1 gene, as may beobtained by isolating the 5′ non-coding sequences located upstream ofthe coding segment or exon, for example, using recombinant cloningand/or PCR™ technology (PCR™ technology is disclosed in U.S. Pat. No.4,683,202 and U.S. Pat. No. 4,682,195, incorporated herein byreference).

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a β2GP1 gene in its natural environment. Suchpromoters may include promoters normally associated with other genes,and/or promoters isolated from any other bacterial, viral, eukaryotic,or mammalian cell, and/or promoters that are not “naturally occurring,”i.e., containing difference elements from different promoters, ormutations that increase, decrease, or alter expression.

Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, tissue, ororganism chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al.(1989), supra, incorporated herein by reference. The promoters employedmay be constitutive, or inducible, and can be used under the appropriateconditions to direct high level expression of the introduced DNAsegment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Promoters of varying strength in termsof their ability to drive expression may also be used to driveexpression of the β2GP1 gene.

At least one module in a promoter generally functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation. Additional promoterelements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 base pairs (bp)upstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. Depending on the promoter, it appears thatindividual elements can function either co-operatively or independentlyto activate transcription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell or tissue is targeted, it is preferable to position the nucleicacid coding region adjacent to and under the control of a promoter thatis capable of being expressed in that human cell or tissue. Generallyspeaking, such a promoter might include either a human or viralpromoter.

Several different types of promoters may be used to drive expression ofβ2GP1, including a promoter from a naturally-occurring heterologousgene, a promoter from an endogenous β2GP1 gene, tissue-specific,developmental pattern, or cell-type-specific promoters, or atranscriptional regulatory element heterogenous with respect to both theβ2GP1 encoding sequences and the host cell or tissue type. Many viralpromoters are appropriate for use in an expression vector or constructto obtain high-level expression of β2GP1 (e.g., retroviral ITRs, LTRs,immediate early viral promoters (IEp) (such as herpesvirus IEp (e.g.,ICP4-IEp and ICP0-IEp), cytomegalovirus (CMV)IEp, and the SV40 earlypromoter), and other viral promoters (e.g., late viral promoters,latency-active promoters (LAPs), Rous Sarcoma Virus (RSV) promoters, andMurine Leukemia Virus (MLV) promoters)). Other suitable promoters areeukaryotic promoters, such as enhancers (e.g., the rabbit β-globinregulatory elements), constitutively active promoters (e.g., the β-actinpromoter, etc.), signal specific promoters (e.g., inducible and/orrepressible promoters, such as a promoter responsive to TNF or RU486,the metallothionine promoter, etc.), and tumor-specific promoters. Theuse of other viral or mammalian cellular or bacterial phage promoterswhich are well-known in the art to achieve expression are contemplatedas well, provided that the levels of expression are sufficient for agiven purpose. This list of is not intended to be exhaustive of all thepossible elements involved in the promotion of expression but, merely,to be exemplary thereof.

The expression or vector construct can also include other geneticelements, for example, polyadenylation sequences, transcriptionalregulatory elements (e.g., enhancers, silencers, etc.), or othersequences. An enhancer is a regulatory DNA sequence that is capable ofactivating transcription from a promoter linked to it. Enhancers canfunction in both orientations either upstream or downstream of apromoter. Some embodiments will employ transcriptional regulatorysequences which confer high level expression and/or a cell-type-specificexpression pattern. Enhancers were originally detected as geneticelements that increased transcription from a promoter located at adistant position on the same molecule of DNA. Subsequent work showedthat regions of DNA with enhancer activity are organized much likepromoters. That is, they are composed of many individual elements, eachof which binds to one or more transcriptional proteins. The basicdistinction between enhancers and promoters is operational. An enhancerregion as a whole must be able to stimulate transcription at a distance;this need not be true of a promoter region or its component elements. Onthe other hand, a promoter must have one or more elements that directinitiation of RNA synthesis at a particular site and in a particularorientation, whereas enhancers lack these specificities. Promoters andenhancers are often overlapping and contiguous, often seeming to have avery similar modular organization.

Additionally, any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression. Use ofa T3, T7, or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

The expression vector or construct may also take advantage of endogenousregulatory elements in the genome of the particular cells or tissue ofinterest, by integrating into a chromosomal location containingfunctional endogenous regulatory elements that are suitable for theexpression of β2GP1 sequences. Endogenous regulatory elements includesequences that are natural to the host cell, tissue, or organism, aswell as sequences present in the host as a result of an infectiousdisease, e.g. virus, prion, and the like. The expression vector orconstruct may integrate into the genome via nonhomologous integration ortargeted homologous recombination, or the expression vector or constructmay express β2GP1 without integrating into the genome.

Expression vectors or constructs contemplated herein can also includeother genetic elements, such as, for example, genes encoding aselectable marker (e.g., β-gal or a marker conferring resistance to atoxin), a pharmacologically active protein, a transcription factor, orother biologically active substance. Positive selection expressioncassettes used in a vector or construct encode a selectable marker thataffords a means for selecting cells that have integrated the vectortransgene sequence spanning the positive selection expression cassette.Suitable drug resistance genes are well known to those of skill in theart and include but are not limited to: gpt (xanthine-guaninephosphoribosyltransferase), which can be selected for with mycophenolicacid; neo (neomycin phosphotransferase), which can be selected for withG418 or hygromycin; and DFHR (dihydrofolate reductase), which can beselected for with methotrexate (Mulligan and Berg, (1981) Proc. Natl.Acad. Sci. USA 78:2072; Southern and Berg, (1982) J. Mol. Appl. Genet.1:327). Positive selection involves expression of the selectable marker,which encodes a functional protein (e.g., neo or gpt) that confers aselectable phenotype to targeted cells or tissue that harbor theendogenously integrated expression cassette, such that by addition of aselection agent (e.g., G418 or mycophenolic acid), targeted cells have agrowth or survival advantage over cells that do not have an integratedexpression cassette.

The β2GP1 coding sequence present in a suitable expression vector orconstruct may be a cDNA sequence, genomic DNA sequence, or the codingsequence may include one or more introns. Many genes or cDNAs thatencode β2GP1 polypeptides are known in the art, or can be deduced fromknown polypeptide sequences. Once a suitable clone or clones of β2GP1coding sequences have been obtained, whether they be cDNA or genomicsequences, an expression system may be prepared. The engineering of DNAsegments for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression.

It is believed that virtually any expression system may be employed inthe expression of the proteins of the present disclosure. Both cDNA andgenomic sequences are suitable for eukaryotic expression, as the hostcell will generally process the genomic transcripts to yield functionalmRNA for translation into protein. Generally speaking, it may be moreconvenient to employ as the recombinant gene a cDNA version of the gene.It is believed that the use of a cDNA version will provide advantages inthat the size of the gene will generally be much smaller and morereadily employed to transfect the targeted cell than will a genomicgene, which will typically be up to an order of magnitude or more largerthan the cDNA gene. However, it is contemplated that a genomic versionof a particular gene may be employed where desired.

In expression, typically a polyadenylation signal is included to effectproper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the disclosure, and any such sequence may be employed.Preferred embodiments include the SV40 polyadenylation signal and thebovine growth hormone polyadenylation signal, convenient and known tofunction well in various target cells. Also contemplated as an elementof the expression cassette is a terminator. These elements can serve toenhance message levels and to minimize read through from the expressioncassette into other sequences.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

It is proposed that β2GP1 proteins, polypeptides, or peptides may beco-expressed with other selected proteins, wherein the proteins may beco-expressed in the same cell or a β2GP1 gene may be provided to a cellthat already expresses another selected protein. Co-expression may beachieved by co-transfecting the cell with two distinct recombinantvectors, each bearing a copy of either of the respective DNA.Alternatively, a single recombinant vector may be constructed to includethe coding regions for both of the proteins, which could then beexpressed in cells transfected with the single vector. In either event,the term “co-expression” herein refers to the expression of both β2GP1and the other selected protein in the same recombinant cell.

As used herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous DNAsegment or gene, such as a cDNA or gene encoding a β2GP1 polypeptide,has been introduced. Therefore, engineered cells are distinguishablefrom naturally occurring cells which do not contain a recombinantlyintroduced exogenous DNA segment or gene. Recombinant cells includethose having an introduced cDNA or genomic gene, and also include genespositioned adjacent to a promoter not naturally associated with theparticular introduced gene. To express a recombinant β2GP1 protein,polypeptide, or peptide, whether mutant or wild-type, in accordance withthe present disclosure, one would prepare an expression vector thatcomprises a wild-type, or mutant β2GP1 protein-encoding nucleic acidunder the control of one or more promoters. In general, to bring acoding sequence under the control of a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe between about 1 and about 50 nucleotides “downstream” of (i.e., 3′of) the chosen promoter. The “upstream” promoter stimulatestranscription of the DNA and promotes expression of the encodedrecombinant protein. This is the meaning of “recombinant expression” inthis context.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein, polypeptide, or peptide expression in a variety ofhost-expression systems. Once a given type of vector or construct isselected, its genome must be manipulated for use as a background vector,after which it must be engineered to incorporate exogenouspolynucleotides. Methods for manipulating the genomes of vectors arewell known in the art (see, e.g., Sambrook et al., supra), and includedirect cloning, site specific recombination using recombinases,homologous recombination, and other suitable methods of constructing arecombinant vector. Vectors containing a targeting construct aretypically grown in E. coli and then isolated using standard molecularbiology methods, or may be synthesized as oligonucleotides.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). In certain embodiments, a cell maycomprise, but is not limited to, at least one skin, bone, neuron, axon,cartilage, blood vessel, cornea, muscle, fascia, brain, prostate,breast, endometrium, lung, pancreas, small intestine, blood, liver,testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymphnode, bone marrow, kidney, peripheral blood, embryonic or ascite cell,and all cancers thereof. An appropriate host can be determined by one ofskill in the art based on the vector backbone and the desired result.

Cell types available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors.Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coliW3110 (F—, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.Additional bacterial cells used as host cells for vector replicationand/or expression include DH5a, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE©, La Jolla). Alternatively, bacterialcells such as E. coli LE392 could be used as host cells for phageviruses. Examples of eukaryotic host cells for replication and/orexpression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, Chinesehamster ovary (CHO), Saos, PC 12, and S2 cells. Many host cells fromvarious cell types and organisms are available and would be known to oneof skill in the art.

Some β2GP1 may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using derivatives of pBR322, a plasmid derived from an E.coli species. pBR322 contains genes for ampicillin and tetracyclineresistance and thus provides easy means for identifying transformedcells. The pBR plasmid, or other microbial plasmid or phage must alsocontain, or be modified to contain, promoters which can be used by themicrobial organism for expression of its own proteins. Additionally,phage vectors containing replicon and control sequences that arecompatible with the host microorganism can be used as transformingvectors in connection with these hosts. For example, the phage lambdaGEM™-11 may be utilized in making a recombinant phage vector which canbe used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors (Inouye et al., 1985); andpGEX vectors, for use in generating glutathione S-transferase (GST)soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with β-galactosidase,ubiquitin, and the like. Promoters that are most commonly used inrecombinant DNA construction include the β-lactamase (penicillinase),lactose and tryptophan (trp) promoter systems. While these are the mostcommonly used, other microbial promoters have been discovered andutilized, and details concerning their nucleotide sequences have beenpublished, enabling those of skill in the art to ligate themfunctionally with plasmid vectors.

The following details concerning recombinant protein production inbacterial cells, such as E. coli, are provided by way of exemplaryinformation on recombinant protein production in general, the adaptationof which to a particular recombinant expression system will be known tothose of skill in the art. Bacterial cells, for example, E. coli,containing the expression vector are grown in any of a number ofsuitable media, for example, LB. The expression of the recombinantprotein may be induced, e.g., by adding IPTG to the media or byswitching incubation to a higher temperature. After culturing thebacteria for a further period, generally of between 2 and 24 h, thecells are collected by centrifugation and washed to remove residualmedia. The bacterial cells are then lysed, for example, by disruption ina cell homogenizer and centrifuged to separate the dense inclusionbodies and cell membranes from the soluble cell components. Thiscentrifugation can be performed under conditions whereby the denseinclusion bodies are selectively enriched by incorporation of sugars,such as sucrose, into the buffer and centrifugation at a selectivespeed.

If the recombinant protein is expressed in the inclusion bodies, as isthe case in many instances, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g.8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents, such as (β-mercaptoethanol or DTT (dithiothreitol).Under some circumstances, it may be advantageous to incubate the proteinfor several hours under conditions suitable for the protein to undergo arefolding process into a conformation which more closely resembles thatof the native protein. Such conditions generally include low proteinconcentrations, for example, less than 500 mg/ml, low levels of reducingagent, concentrations of urea less than 2 M and often the presence ofreagents such as a mixture of reduced and oxidized glutathione whichfacilitate the interchange of disulfide bonds within the proteinmolecule. The refolding process can be monitored, for example, bySDS-PAGE, or with antibodies specific for the native molecule (which canbe obtained from animals vaccinated with the native molecule or smallerquantities of recombinant protein). Following refolding, the protein canthen be purified further and separated from the refolding mixture bychromatography on any of several supports including ion exchange resins,gel permeation resins, or on a variety of affinity columns.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used. This plasmid already contains the trp1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. Thepresence of the trp1 lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Suitablepromoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other suitable promoters, which have the additionaladvantage of transcription controlled by growth conditions, include thepromoter region for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived frommulticellular organisms may also be used as hosts, and are known tothose of skill in the art. In principle, any such cell culture isworkable, whether from vertebrate or invertebrate culture. In additionto mammalian cells, these include insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus); and plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing one or more β2GP1 protein, polypeptide, or peptidecoding sequences. Examples of useful mammalian host cell lines are VEROand HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK,COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines. Additionally, a hostcell strain may be chosen that modulates the expression of the insertedsequences, or modifies and processes the gene product in the specificfashion desired. Such modifications (e.g., glycosylation) and processing(e.g., cleavage) of protein products may be important for the functionof the protein. Different host cells have characteristic and specificmechanisms for the post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed.

Expression vectors for use in mammalian cells ordinarily include anorigin of replication (as necessary), a promoter located in front of thegene to be expressed, along with any necessary ribosome binding sites,RNA splice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)sources, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient. The promoters may be derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Further, it is also possible, and may bedesirable, to utilize promoter or control sequences normally associatedwith β2GP1 gene sequence(s), provided such control sequences arecompatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2, and mostfrequently Simian Virus 40 (SV40). The early and late promoters of SV40virus are particularly useful because both are obtained easily from thevirus as a fragment, which also contains the SV40 viral origin ofreplication. In cases where an adenovirus is used as an expressionvector, the coding sequences may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1, E3, or E4)will result in a recombinant virus that is viable and capable ofexpressing β2GP1 proteins, polypeptides, or peptides in infected hosts.

Specific initiation signals may also be required for efficienttranslation of β2GP1 protein, polypeptide, or peptide coding sequences.These signals include the ATG initiation codon and adjacent sequences.Exogenous translational control signals, including the ATG initiationcodon, may also need to be provided. One of ordinary skill in the artwould readily be capable of determining this and providing the necessarysignals. It is well known that the initiation codon must be in-frame (orin-phase) with the reading frame of the desired coding sequence toensure translation of the entire insert. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements andtranscription terminators. In eukaryotic expression, one will alsotypically desire to incorporate into the transcriptional unit anappropriate polyadenylation site (e.g., 5′-AATAAA-3′), particularly ifone is not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

For long-term, high-yield production of a recombinant β2GP1 protein,polypeptide, or peptide, stable expression is preferred. For example,cell lines that stably express constructs encoding a β2GP1 protein,polypeptide, or peptide may be engineered. Rather than using expressionvectors or constructs that contain viral origins of replication, hostcells can be transformed with vectors controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of foreign DNA, engineeredcells are allowed to grow for 1-2 days in an enriched media, and thenare switched to a selective media. The selectable marker in therecombinant plasmid confers resistance to the selection and allows cellsto stably integrate the plasmid into their chromosomes and grow to formfoci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (tk), hypoxanthine-guaninephosphoribosyltransferase (hgprt) and adenine phosphoribosyltransferase(aprt) genes, in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordihydrofolate reductase (dhfr), that confers resistance to methotrexate;gpt, that confers resistance to mycophenolic acid; neomycin (neo), thatconfers resistance to the aminoglycoside G-418; and hygromycin (hygro),that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. Suspension cultured cells havelimitations, however, such as tumorigenic potential and lower proteinproduction than adherent cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts. The airlift reactor, also initially described for microbialfermentation and later adapted for mammalian culture, relies on a gasstream to both mix and oxygenate the culture. The gas stream enters ariser section of the reactor and drives circulation. Gas disengages atthe culture surface, causing denser liquid free of gas bubbles to traveldownward in the downcomer section of the reactor. The main advantage ofthis design is the simplicity and lack of need for mechanical mixing.Typically, the height-to-diameter ratio is 10:1. The airlift reactorscales up relatively easily, has good mass transfer of gases, andgenerates relatively low shear forces.

In order to effect expression of gene constructs, the expressionconstruct must be delivered into a cell. Viral vectors are besttransferred into the cells by infecting them; however, the mode ofinfection can vary depending on the virus. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo (see below), as in the treatment ofcertain disease states. The preferred mechanism for delivery is viaviral infection where the expression construct is encapsidated in aninfectious viral particle. Several non-viral methods for the transfer ofexpression constructs into cultured mammalian cells are alsocontemplated by the present disclosure. Expression vectors or constructscan be introduced or transferred to cells or tissues of interest by anyone of many known suitable techniques, including but not limited tomicroinjection, biolistics, calcium phosphate precipitation (Graham andVan Der Eb, Virology 54:536-539, 1973; Chen and Okayama, Mol. Cell Biol.7:2745-2752, 1987; Rippe et al., Mol. Cell. Biol. 10:689-695, 1990,incorporated herein by reference) DEAE-dextran (Gopal, Mol. Cell Biol.5:1188-1190, 1985, incorporated herein by reference), electroporation(Tur-Kaspa et al., Mol. Cell Biol. 6:716-718, 1986; Potter et al., Proc.Natl. Acad. Sci. (USA) 81:7161-7165, 1984, incorporated herein byreference), direct microinjection (Harland and Weintraub, J. Cell Biol.101:1094-1099, 1985, incorporated herein by reference), DNA-loadedliposomes (Nicolau and Sene, Biochim. Biophys. Acta 721:185-190, 1982;Fraley et al., J. Biol. Chem. 255:10431-10435, 1980, incorporated hereinby reference) and lipofectamine-DNA complexes, cell sonication(Fechheimer et al., Proc. Natl. Acad. Sci. (USA) 84:8463-8467, 1987,incorporated herein by reference), gene bombardment using high velocitymicroprojectiles (Fitzpatrick-McElligott, Biotechnology 10:1036-1040,1992, incorporated herein by reference), receptor-mediated transfection(Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988; Wu and Wu,Biochemistry 27:887-892, 1988, incorporated herein by reference), genegun, and others (see, generally, Sambrook et al., supra; see also Watsonet al., (1992) Recombinant DNA, Chapter 12, 2d edition, ScientificAmerican Books; incorporated herein by reference). Other methods used totransform mammalian cells include the use of polybrene-mediated transferand protoplast fusion. Some of these techniques may be successfullyadapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably maintained in the cell as a separate,episomal segment of DNA. Such nucleic acid segments or “episomes” encodesequences sufficient to permit maintenance and replication independentof or in synchronization with the host cell cycle. How the expressionconstruct is delivered to a cell and where in the cell the nucleic acidremains is dependent on the type of expression construct employed.

In one embodiment of the present disclosure, the expression vector orconstruct may simply consist of naked recombinant DNA or plasmids.Transfer of the construct may be performed by any of the methodsmentioned above which physically or chemically permeabilize the cellmembrane. This is particularly applicable for transfer in vitro but itmay be applied to in vivo use as well. Dubensky et al. (Proc. Natl.Acad. Sci. (USA) 81:7529-7533, 1984) successfully injected polyomavirusDNA in the form of CaPO₄ precipitates into liver and spleen of adult andnewborn mice demonstrating active viral replication and acute infection.Benvenisty and Reshef (Proc. Natl. Acad. Sci. (USA) 83:9551-9555, 1986,incorporated herein by reference) also demonstrated that directintraperitoneal injection of CaPO₄ precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest may also be transferred in a similar manner in vivoand express the gene product.

Another embodiment of the disclosure for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., Biotechnology 10:286-291,1992, incorporated herein by reference). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force. The microprojectiles used have consistedof biologically inert substances such as tungsten or gold beads.Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Zelenin et al., Genetika 27:2182-2186,1991, incorporated herein by reference). This may require surgicalexposure of the tissue or cells, to eliminate any intervening tissuebetween the gun and the target organ, i.e., ex vivo treatment.

In a further embodiment of the disclosure, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of dosed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, Targeted Diagn. Ther. 4:87-103, 1991). Also contemplated arelipofectamine-DNA complexes. Liposome-mediated nucleic acid delivery andexpression of foreign DNA in vitro has been very successful. Wong et al.(Gene 10:87-94, 1980, incorporated herein by reference) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al.(Methods Enzymol. 149:157-176, 1987, incorporated herein by reference)accomplished successful liposome-mediated gene transfer in rats afterintravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., J. Biol. Chem.264:12126-12129, 1989, incorporated herein by reference). In otherembodiments, the liposome may be complexed or employed in conjunctionwith nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., J.Biol. Chem. 266:3361-3364, 1991, incorporated herein by reference). Inyet further embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentdisclosure. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs that can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific. Receptor-mediated genetargeting vehicles generally consist of two components: a cellreceptor-specific ligand and a DNA-binding agent. Several ligands havebeen used for receptor-mediated gene transfer. The most extensivelycharacterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, J. Biol.Chem. 262:4429-4432, 1987, incorporated herein by reference) andtransferrin (Wagner et al., Proc. Natl. Acad. Sci. (USA) 87:3410-3414,1990, incorporated herein by reference). A synthetic neoglycoprotein,which recognizes the same receptor as ASOR, has also been used as a genedelivery vehicle (Ferkol et al., FASEB 7:1081-1091, 1993; Perales etal., Proc. Natl. Acad. Sci. (USA) 91:4086-4090, 1994, incorporatedherein by reference) and epidermal growth factor (EGF) has been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In an embodiment of the present disclosure, β2GP1 may be administered inthe form of a chimeric protein comprising the biologically activeportion of β2GP1 and a ligand. The ligand may be a growth factor,chemokine, growth factor receptor, antibody and the like that targetsβ2GP1 to a specific site, protein, or cell type. Administration of theβ2GP1 chimeric protein allows for efficient targeting of β2GP1 to asite. For example, the ligand comprising the chimeric protein may be amodified bFGF protein that binds to the FGF receptor but does not causeangiogenesis. In another example, the chimeric protein may include aligand for the endothelial cell surface molecule CD31 or otherendothelial cell ligands.

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (Methods Enzymol. 149:157-176,1987, incorporated herein by reference) employed lactosyl-ceramide, agalactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a particular gene alsomay be specifically delivered into a cell type such as endothelial ortumor cells, by any number of receptor-ligand systems with or withoutliposomes. For example, epidermal growth factor (EGF) may be used as thereceptor for mediated delivery of a nucleic acid encoding a gene in manytumor cells that exhibit upregulation of EGF receptor. Also, antibodiesto CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia), and MAA(melanoma) can similarly be used as targeting moieties.

Cells into which the β2GP1 gene has been transferred can be used in thepresent disclosure as transient transformants. Alternatively, where thecells are cells in vitro, they can be subjected to several rounds ofclonal selection (if the vector also contains a gene encoding aselectable marker, such as a gene conferring resistance to a toxin) toselect for stable transformants. Within the cells, the β2GP1 gene isexpressed such that the cells express the β2GP1 polypeptide. Successfulexpression of the gene can be assessed via standard molecular biologicaltechniques (e.g., Northern hybridization, Western blotting,immunoprecipitation, enzyme immunoassay, etc.).

D. In Vivo Delivery and Treatment Protocols

The present disclosure also encompasses methods for the in vivo or exvivo delivery of β2GP1, for example to treat a tumor or a patient usinggene therapy. Many expression vectors known in the art are particularlywell suited for in vivo delivery of nucleic acid sequences that are thenexpressed in cells, tissues, or organisms of interest, for exampleadenovirus, retrovirus, vaccinia virus, adeno-associated virus (AAV),herpes viruses, and hepatitis B virus. In certain embodiments, genetransfer may be more easily be performed under ex vivo conditions. Exvivo gene therapy refers to the isolation of cells from an organism, thedelivery of a nucleic acid into the cells, in vitro, and then the returnof the modified cells back into the organism. This may involve thesurgical removal of tissue/organs from an organism or the primaryculture of cells and tissues. Anderson et al., U.S. Pat. No. 5,399,346,incorporated herein in its entirety, discloses ex vivo therapeuticmethods.

(i) Adenovirus

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. As used herein, “adenovirus expressionvector” refers to those constructs containing adenovirus sequencessufficient to (a) support packaging of the construct and (b) to expressa polynucleotide that has been cloned therein. In this context,expression does not require that the gene product be synthesized, but ina preferred embodiment, the gene product is expressed.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization or adenovirus, a 36kilobases (kB), linear, double-strained DNA virus, allows substitutionof large pieces of adenoviral DNA with foreign sequences up toapproximately 7 kB. In contrast to retrovirus, the infection ofadenovira DNA in host cells does not result in chromosomal integrationbecause adenoviral DNA can replicate in an episomal manner withoutpotential genotoxicity. Also, adenoviruses are structurally stable, andno genome rearrangement has been detected after extensive amplification.So far, adenoviral infection appears to be linked only to mild diseasesuch as acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its midsized genome, ease of manipulation, high titer, widetarget-cell range and high infectivity. In a current system, recombinantadenovirus is generated from homologous recombination between shuttlevector and provirus vector. Due to the possible recombination betweentwo proviral vectors, wild-type adenovirus may be generated from thisprocess. Therefore, it is critical to isolate a single clone of virusfrom an individual plaque and examine its genomic structure. Use of theYAC system is an alternative approach for the production of recombinantadenovirus.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, for example,the helper cell line designated 293 (Graham et al., J. Gen. Virol.36:59-74, 1977, incorporated herein by reference). Helper cell lines maybe derived from human cells such as human embryonic kidney cells, musclecells, hematopoietic cells, or other human embryonic mesenchymal orepithelial cells. Alternatively, the helper cells may be derived fromthe cells of other mammalian species that are permissive for humanadenovirus. Such cells include, e.g., Vero cells or other monkeyembryonic mesenchymal or epithelial cells. As stated above, thepreferred helper cell line is 293.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the disclosure. The adenovirus may be one of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent disclosure. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector. The typical vectoraccording to the present disclosure is replication defective and doesnot have an adenovirus E1 region. Thus, it will be most convenient tointroduce the polynucleotide encoding the gene of interest at theposition where the E1 coding sequences have been removed. However, theposition of insertion of the construct within the adenovirus sequencesis not critical. The polynucleotide encoding the gene of interest mayalso be inserted in lieu of the deleted E3 region in E3 replacementvectors or in the E4 region where a helper cell line or helper viruscomplements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits abroad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus, demonstrating their safety and therapeuticpotential as in vivo gene transfer vectors.

Adendovirus have been used in eukaryotic gene expression and vaccinedevelopment. Recently, animal studies suggested that recombinantadenovirus could be used for gene therapy (Stratford-Perricaudet et al.,Bone Marrow Transplant 9 Suppl. 1:151-152, 1992; Stratford-Perricaudetet al., Hum. Gene Ther. 1:241-256, 1990; Rich et al., Hum. Gene Ther.4:461-476, 1993, incorporated herein by reference). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., Science 252:431-434, 1991;Voshimura et al., Nucleic Acids Res. 20:3233-3240, 1992, incorporatedherein by reference), muscle injection (Ragot et al., Nature361:647-650, 1993, incorporated herein by reference), peripheralintravenous injections (Herz and Gerard, Proc. Natl. Acad. Sci. (USA)90:2812-2816, 1993, incorporated herein by reference) and stereotaticinoculation into the brain (Le Gal La Salle et al., Science 259:988-990,1993, incorporated herein by reference).

(ii) Retroviruses

Retroviruses are a group of single-stranded RNA viruses characterized byan ability to convert their RNA to double-stranded DNA to infected cellsby a process of reverse-transcription. The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively.Methods for packaging a recombinant plasmid containing a nucleic acidsequence of interest, which are then secreted into the culture media,are well known in the art (see, e.g., Nicolas and Rubenstein,Biotechnology 10:493-513, 1988, incorporated herein by reference). Themedia containing the recombinant retroviruses is collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., Virology67:242-248, 1975).

There are certain limitations to the use of retrovirus vectors in allaspects of the present disclosure. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes. Another concern with the use ofdefective retrovirus vectors is the potential appearance of wild-typereplication-competent virus in the packaging cells. New packaging celllines are now available, however, that should greatly decrease thelikelihood of recombination (see, e.g., Markowitz et al., Adv. Exp. Med.Biol. 241:35-40, 1988, incorporated herein by reference).

(iii) Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in thepresent disclosure. Vectors derived from viruses such as vaccinia virus(Coupar et al., Gene 68:1-10, 1988, incorporated, herein by reference),adeno-associated virus (AAV) (Hermonat and Muzycska, Proc. Natl. Acad.Sci. (USA) 81:6466-6470, 1984, incorporated herein by reference), herpesviruses, and defective hepatitis B virus (Chang et al., J. Viral.68:646-653, 1991, incorporated herein by reference) may be employed.These viral vectors offer several attractive features for variousmammalian cells.

E. Pharmaceutical Compositions and Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Pharmaceutical compositions disclosed herein canbe administered to a subject using a variety of routes of administrationand dosage forms well known to those of skill in the art. As usedherein, “pharmaceutical compositions of β2GP1” include compositions ofβ2GP1 polypeptides produced from natural, recombinant, or syntheticsources; expression vectors or constructs encoding β2GP1, includingviral and non-viral gene delivery vectors or construct; and engineeredcells expressing recombinant β2GP1. Generally, this will entailpreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to cells, humans, or animals.

Pharmaceutical compositions of β2GP1 also include proteinaceouscomposition, which encompasses β2GP1 proteins, polypeptides, andpeptides. In certain embodiments, the present disclosure concernsproteinaceous compositions comprising at least one proteinaceousmolecule. As used herein, a “proteinaceous molecule” or “proteinaceouscomposition” generally refers, but is not limited to, a protein ofgreater than about 200 amino acids or the full length endogenoussequence translated from a gene, for example, a β2GP1 gene; apolypeptide of greater than about 100 amino acids; and/or a peptide offrom about 3 to about 100 amino acids. “Proteinaceous” terms describedabove may be used interchangably herein.

In certain embodiments the size of the at least one proteinaceousmolecule may comprise, but is not limited to, about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, about 20, about 21, about 22, about 23, about 24, about25, about 26, about 27, about 28, about 29, about 30, about 31, about32, about 33, about 34, about 35, about 36, about 37, about 38, about39, about 40, about 41, about 42, about 43, about 44, about 45, about46, about 47, about 48, about 49, about 50, about 51, about 52, about53, about 54, about 55, about 56, about 57, about 58, about 59, about60, about 61, about 62, about 63, about 64, about 65, about 66, about67, about 68, about 69, about 70, about 71, about 72, about 73, about74, about 75, about 76, about 77, about 78, about 79, about 80, about81, about 82, about 83, about 84, about 85, about 86, about 87, about88, about 89, about 90, about 91, about 92, about 93, about 94, about95, about 96, about 97, about 98, about 99, about 100, about 110, about120, about 130, about 140, about 150, about 160, about 170, about 180,about 190, about 200, about 210, about 220, about 230, about 240, about250, about 275, about 300, about 325, about 350, about 375, about 400,about 425, about 450, about 475, about 500, about 525, about 550, about575, about 600, about 625, about 650, about 675, about 700, about 725,about 750, about 775, about 800, about 825, about 850, about 875, about900, about 925, about 950, about 975, about 1000, about 1100, about1200, about 1300, about 1400, about 1500, about 1750, about 2000, about2250, about 2500 or greater amino molecule residues, and any rangederivable therein.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative, or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties. Accordingly, a proteinaceous composition encompassesamino molecule sequences comprising at least one of the 20 common aminoacids in naturally synthesized proteins, or at least one modified orunusual amino acid, known to those of skill in the art.

In certain embodiments, the proteinaceous composition comprises at leastone protein, polypeptide, or peptide. In further embodiments, theproteinaceous composition comprises a biocompatible protein,polypeptide, or peptide. As used herein, the term “biocompatible” refersto a substance which produces no significant untoward effects whenapplied to, or administered to, a given organism according to themethods and amounts described herein. Such untoward or undesirableeffects are those such as significant toxicity or adverse immunologicalreactions. In preferred embodiments, biocompatible protein, polypeptideor peptide containing compositions will generally be mammalian proteinsor peptides or synthetic proteins or peptides each essentially free fromtoxins, pathogens, and harmful immunogens.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptides,or peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide, and peptide sequences for various β2GP1 genes havebeen previously disclosed, and may be found at computerized databasesknown to those of ordinary skill in the art. One such database is theNational Center for Biotechnology Information's Genbank and GenPeptdatabases (http://www.ncbi.nlm.nih.gov/). The coding regions for theseknown genes may be amplified and/or expressed using the techniquesdisclosed herein or as would be know to those of ordinary skill in theart. Alternatively, various commercial preparations of proteins,polypeptides, and peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a protein, polypeptide, or peptidecomposition that has been subjected to fractionation to remove variousother proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, byprotein assays, as would be known to one of ordinary skill in the artfor the specific or desired protein, polypeptide, or peptide.

In certain embodiments, the proteinaceous composition may comprise atleast one antibody. It is contemplated that antibodies to specifictissues may bind the tissue(s) and foster tighter adhesion to thetissue(s). As used herein, the term “antibody” is intended to referbroadly to any immunologic binding agent such as IgG, IgM, IgA, IgD andIgE. Generally, IgG and/or IgM are preferred because they are the mostcommon antibodies in the physiological situation and because they aremost easily made in a laboratory setting. The term “antibody” is used torefer to any antibody-like molecule that has an antigen binding region,and includes antibody fragments such as Fab′, Fab, F(ab′)2, singledomain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Thetechniques for preparing and using various antibody-based constructs andfragments are well known in the art. Means for preparing andcharacterizing antibodies are also well known in the art (See, e.g.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;incorporated herein by reference).

It is contemplated that virtually any protein, polypeptide, or peptidecontaining component may be used in the compositions and methodsdisclosed herein. In preferred embodiments, however, the proteinaceousmaterial is biocompatible. In certain embodiments, it is envisioned thatthe formation of a more viscous composition will be advantageous in thatwill allow the composition to be more precisely or easily applied to thetissue and to be maintained in contact with the tissue throughout theprocedure. In such cases, the use of a peptide composition, or morepreferably, a polypeptide or protein composition, is contemplated.Ranges of viscosity include, but are not limited to, about 40 to about100 poise. In certain aspects, a viscosity of about 80 to about 100poise is preferred.

Polypeptides, proteins, and peptides suitable for use in the presentdisclosure may be autologous proteins or peptides, although thedisclosure is clearly not limited to the use of such autologousproteins. As used herein, the term “autologous protein, polypeptide, orpeptide” refers to a protein, polypeptide or peptide which is derived orobtained from an organism. Organisms that may be used include, but arenot limited to, a primate, a canine, a feline, a bovine, an equine, anovine, a murine, a caprine, a porcine species, and the like, with aselected animal or human subject being preferred. The “autologousprotein, polypeptide, or peptide” may then be used as a component of acomposition intended for application to the selected animal or humansubject.

To select other proteins, polypeptides, peptides and the like for use inthe methods and compositions of the present disclosure, one wouldpreferably select a proteinaceous material that possesses one or more ofthe following characteristics: it forms a solution with a highpercentage of proteinaceous material solubilized; it possesses a highviscosity (i.e. about 40 to about 100 poise); it has the correctamino-acids present to form covalent cross-links; and/or it isbiocompatible (i.e. from mammalian origin for mammals, preferably fromhuman origin for humans, from canine origin for canines, etc.; it isautologous; it is non-allergenic, and/or it is nonimmunogenic).

Pharmaceutical compositions of β2GP1 may be administered through anumber of different routes, including oral, peroral, enteral, pulmonary,rectal, nasal, vaginal, lingual, direct injection, intravenous,intraarterial, intracardial, intradermal, intramuscular,intraperitoneal, intracutaneous, intraocular, intranasal, intrapleural,intrathecal, intratumor, intrauterine, orthotopic, and subcutaneousadministration. β2GP1 may also be suitable for systemic administrationto the subject, including parenteral, topical, buccal, sublingual,transdermal, gavage, and oral administration. β2GP1 may also beadministered parenterally, i.e. subcutaneously, intramuscularly, orintravenously. Agents can also be delivered to a tissue or organism in avariety of different compositions, including tablets, pills, capsules,powders, aerosols, suppositories, skin patches, parenterals, and oralliquids, including oil-aqueous suspensions, solutions, and emulsions.Also contemplated is the administration of β2GP1 in single or multipledosage regimens, as well as by using compositions that involve sustainedrelease (long acting) formulations and devices.

In addition to other routes of administration, β2GP1 can be presented tothe tissue of interest by direct infusion or by using expression vectorsor constructs, including but not limited to genetically modified cellsor tissues, viral vectors, or infusion of genetic material, which canthen be incorporated by cells or in the tissue of a host organism. Thus,for example, a composition containing a source of β2GP1 (i.e., a β2GP1polypeptide or a β2GP1 expression vector or construct, as describedherein) can be introduced into the systemic circulation, which willdistribute the source of β2GP1 to the tissue of interest. Alternatively,a composition containing a source of β2GP1 can be applied topically orinjected directly into a tissue of interest (e.g., injected as a boluswithin a tumor or intercutaneous or subcutaneous site, applied to all ora portion of the surface of the skin, dropped onto the surface of theeye, etc.). Additionally, β2GP1 may be incorporated into biodegradablepolymers that allow for the sustained release of the compound. Thepolymers can be implanted in the vicinity of where drug delivery isdesired, for example, at the site of a tumor, or implanted so that theβ2GP1 is slowly released systemically. The biodegradable polymers andtheir use are described, for example, in Brem et al., J. Neurosurg.74:441-446, 1991, which is incorporated herein by reference.

In general, appropriate salts and buffers will be employed to renderdelivery vectors stable and allow for uptake by target cells. Buffersalso will be employed when recombinant cells are introduced into a cell,tissue, or organism. Aqueous compositions of the present disclosurecontain an effective amount of the expression vector or cells, dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions also are referred to as inocula. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an organism, animal, orhuman. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentdisclosure, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

β2GP1 can be administered as a free base, as well as any acid additionsalt thereof. Pharmacologically acceptable salts of β2GP1 include thosederived from organic and inorganic acids such as, without limitation,hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid,methanesulphonic acid, acetic acid, tartaric acid, lactic acid, succinicacid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid,salicylic acid, phthalic acid, embonic acid, enanthic acid, and thelike. Solutions of the active ingredients as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with surfactant, such as hydroxypropylcellulose. Dispersions alsocan be prepared in glycerol, liquid polyethylene glycols, mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent growth ofmicroorganisms.

The expression vectors, constructs, and delivery vehicles of the presentdisclosure may include classic pharmaceutical preparations.Administration of these compositions according to the present disclosurewill be via any common route so long as the target tissue is availablevia that route. Such compositions would normally be administered aspharmaceutically acceptable compositions, described supra.

The polypeptides, vectors, or cells of the present disclosure areadvantageously administered in the form of injectable compositionseither as liquid solutions or suspensions; solid forms or lyophilizedforms suitable for solution in, or suspension in, liquid prior toinjection also may be prepared. These preparations also may beemulsified. A typical compositions for such purposes comprises a 50 mgor up to about 100 mg of human serum albumin per milliliter of phosphatebuffered saline. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oil, and injectable organicesters, such as theyloleate. Aqueous carriers include water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, etc. Intravenous vehicles includefluid and nutrient replenishers. Preservatives include antimicrobialagents, anti-oxidants, chelating agents and inert gases. The pH andexact concentration of the various components in the pharmaceuticalcompositions are adjusted according to well known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations, nasal sprays, suppositories,tonics, or powders. When the route is topical, the form may be a cream,ointment, salve, gel, or spray.

An effective amount of the therapeutic agent is determined based on theintended goal. For example, an effective amount of β2GP1 as used hereinis an amount of β2GP1 that is effective for inhibiting, treating, orpreventing unwanted endothelial cell proliferation, cord formation, cellmigration, angiogenesis, and/or angioectasia, especially as related toneoplasm or tumor growth. The term “unit dose” refers to a physicallydiscrete unit suitable for use in an organism, each unit containing apredetermined quantity of the therapeutic composition calculated toproduce the desired response in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the organism to be treated, the state of the organism, andthe protection desired. Precise amounts of the therapeutic compositionalso depend on the judgment of the practitioner and may be peculiar toeach individual organism. The optimal daily dose of β2GP1 useful for thepurposes of the present disclosure is determined by methods known in theart, e.g., based on the severity of the angiogenic disease or conditionand the symptoms being treated, the condition of the organism to whomtreatment is being given, the desired degree of therapeutic response,and the concomitant therapies being administered to the organism orhuman. The total daily dosage administered to an organism, typically ahuman patient, should be at least the amount required to inhibit, treat,or prevent unwanted endothelial cell proliferation and/or angiogenesis.

The optimal dosage of administered β2GP1 will be determined by methodsknown in the art and will vary depending on such factors as theorganism's age, weight, height, sex, general medical/clinical condition,previous medical history, disease progression, tumor burden, route ofadministration, formulation, concomitant therapies being administered,observed response of the organism, and the like. The dosage can beadministered in a single or multiple dosage regimen, or delivered in anessentially continuous manner, e.g., via a transdermal patch. Dependingon the formulation of a composition comprising β2GP1, it is suppliedover a time course sufficient to inhibit, treat, prevent, attenuate, orretard unwanted endothelial cell proliferation and/or angiogenesiswithin the desired tissue or organism. In some protocols (e.g., wherethe β2GP1 is supplied to the surface of skin), repeated application mayenhance the antiangiogenic effect. Where the source of β2GP1 is a β2GP1expression vector or construct, cells expressing the vector or constructmay produce an effective amount of the protein (i.e., sufficient toinhibit angiogenesis in the tissue).

In general, it is desirable to provide the organism with a dosage ofβ2GP1 of at least about 0.01 mg/kg, 0.02 mg/kg or 0.05 mg/kg to about0.10 mg/kg, or about 0.15 mg/kg to about 0.175 mg/kg, or about 0.20mg/kg to about 0.30 mg/kg, or about 0.5 mg/kg and may extend to about1.0 mg/kg or even 1.5, 2.0, 3.0, 5.0 or 10.0 mg/kg of the organism'sbody weight depending on the route of administration. In other preferredembodiments, a range of from about 1 mg/kg to about 25 mg/kg, preferablyat least about 50 mg/kg, more preferably about 100 mg/kg iscontemplated, although a lower or higher dose may be administered. Inother preferred embodiments, the daily dose will be in the range ofabout 0.01 mg to about 1000 mg per day. Preferred doses will be about0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or1000 mg per day.

The present disclosure also contemplates providing to a subject apharmacological composition with a source of β2GP1 and a suitablediluent, such as saline, phosphate-buffered saline, or otherphysiologically tolerable diluents. In addition to the source of β2GP1,the composition includes a diluent, which includes one or morepharmacologically-acceptable carriers. Pharmaceutical compositions foruse in accordance with the present disclosure can be formulated in aconventional manner using one or more pharmacologically orphysiologically acceptable carriers comprising excipients, as well asoptional auxiliaries which facilitate processing of the active compoundsinto preparations which can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen. Thus, for systemicinjection, the source of β2GP1 can be formulated in aqueous solutions,preferably in physiologically compatible buffers. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart. For oral administration, the source of β2GP1 can be combined withcarriers suitable for inclusion into tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, diposomes, suspensions and the like.For administration by inhalation, the source of β2GP1 is convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant. The sourceof β2GP1 can be formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Such compositions cantake such forms as suspensions, solutions, or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing. and/or dispersing agents. For application to the skin, thesource of β2GP1 can be formulated into a suitable gel, magma, creme,ointment, or other carrier. For application to the eyes, the source ofβ2GP1 can be formulated in aqueous solutions, preferably inphysiologically compatible buffers. The source of β2GP1 can also beformulated into other pharmaceutical compositions such as those known inthe art.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Quantitative Neovascularization Assays

β2GP1 is able to abrogate neovascularization into subcutaneous gel foamimplants using a novel technique for quantifying vascular volume. Thistechnique uses a reproducible and readily quantifiable assay to studyangiogenesis in vivo, and in particular assesses the propensity ofdifferent compounds to inhibit angiogenesis. This assay uses absorbentsurgical gel foam (Nabai et al., Dermatology 191:240-241, 1995,incorporated herein by reference). Briefly, sterile gel foam absorbablesponges (Pharmacia & Upjohn, NJ) were cut into approximately 5×5 mmpieces and hydrated overnight at 4° C. in sterile phosphate-bufferedsaline (PBS). Excess PBS was removed by blotting. Sterile 0.4% agarose(100 μl) containing human serum albumin, intact β2GP1, or N-β2GP1 wasaliquoted to each sponge cube at 45° C. The gel foam sponges wereallowed to harden for 1 hour at room temperature before implantation.After the mice were anesthetized, a ˜5 mm midline incision was madethrough the skin and subcutaneous pockets were formed on both sides withforceps. One gel foam sponge was inserted into each pocket and the woundwas closed with two surgical metal clips. Fourteen days later, the micewere sacrificed and the gel foam sponges were recovered.

The extent of angiogenesis into the gel foam plugs can be determined byassessing microvessel density (MVD) by immunofluorescence ofantigen-specific endothelial cell markers and by directly estimatingvascular volume (QVV). To quantify the degree ofneovascularization/vascular volume in the gel foam implants recoveredfrom the mice, vascular volume was estimated by assessing blood volumein the gel foam plugs. Mice were first injected intravenously with 0.1mL of 40% packed syngeneic red blood cells (RBC) labeled with ⁵¹Cr (1.0mCi/mL packed red cells). The mice were bled from the tail veinapproximately 10 minutes later and an aliquot (10 μL) of blood wascollected to determine cpm/μL blood. The mice were then sacrificed andthe vascular volume of the excised gel foam implants was quantified byscintillation counting. Vascular volume was determined by comparingcounts from tail vein samples to counts obtained in the plugs (used tocalculate the vascular volume/gram). This same method can also be usedto assess vascular volume and the extent of angiogenesis in tumors.

After vascular volume has been quantified, MVD can be assessed byimmunofluorescence of antigen-specific endothelial cell markers, forexample CD31 and PECAM-1. To do this, frozen gel foam specimens aresectioned (10-12 μm), mounted on positively charged slides, air-driedfor 30 minutes, fixed in cold acetone for 5 minutes, followed byacetone:chloroform (1:1) for 5 minutes, and acetone alone for anadditional 5 minutes. After rehydrating the sections with PBS they areincubated for 20 minutes at room temperature with a protein-blockingsolution containing 5% normal horse serum and 1% normal goat serum inPBS, and then incubated at 4° C. with a 1:400 dilution of rat monoclonalanti-mouse CD31 antibody (Pharmingen, San Diego, Calif.). The slides arethen washed and stained with 1:200 dilution of secondary goat anti-ratantibody conjugated to Texas Red (Jackson Research Laboratories, WestGrove, Calif.). The samples are mounted in Vectashield mounting mediumfor fluorescence with DAPI (Vector Laboratories, Burlingame, Calif.).Fluorescence microscopy can be done with a Zeiss Axioplan2 microscope(Carl Zeiss, New York, N.Y.) equipped with a 100-W HBO mercury bulb andfilter sets to individually capture red, green, and blue fluorescentimages. Images can be captured using a C5810 Hamamatsu color chilled3CCD camera (Hamamatsu, Japan) and digitized using Optimas imagingsoftware (Silver Springs, Md.). Endothelial cells are identified by redfluorescence. For quantification of MVD, ten 0.159-mm² fields at 100×magnification are digitized and stored for analysis.

FIG. 1 shows dramatic neovascularization into control implants (gel foamplug A). Plugs containing intact β2GP1 or N-β2GP1, on the other hand,appear to completely block the growth of new blood vessels into theimplants. Moreover, control plugs (containing either buffer alone orhuman serum albumin) were strongly attached to the fascia whereas mostof the β2GP1-containing plugs remained very loosely attached or notattached at all (FIG. 1). The extent of vascularization into theimplants quantified by scintillation counting indicated that neovascularization was inhibited >10-fold in plugs containing β2GP1 (FIG.2). Note that both intact β2GP1 and N-β2GP1 inhibited neovascularizationinto the plugs. It is possible that since implantation of the gel foamcreates a wound, intact β2GP1 may be cleaved to the nicked form byendogenous proteases in situ. Similarly, wounding caused by subcutaneousinjection could also result in activation of endogenous plasmin andcleavage of the injected β2GP1 to N-β2GP1.

Example 2 β2GP1 Kills and Inhibits the Growth of In Vitro CultivatedPulmonary Microvascular Endothelial Cells

The inhibitory effect of β2GP1 on neovascularization shown in Example 1could be due to a variety of effects on endothelial cells, including butnot limited to β2GP1-induced apoptosis, inhibition of cell growth, cellmigration, and/or cord assembly. To determine whether intact β2GP1and/or N-β2GP1 inhibit cell growth or induce apoptosis, murine pulmonarymicrovascular endothelial cells were grown for 4 days in the presence ofβ2GP1, N-β2GP1 and, as a control, human serum albumin (Log₂ dilutionfrom 100 μg/ml). An MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure the cell growth of each set oftreated pulmonary microvascular cells.

The data in the left panel of FIG. 3 shows a substantial reduction inthe number of viable endothelial cells after 4 days of incubation withintact β2GP1 and N-β2GP1. Importantly, N-β2GP1 was ˜20 times moreeffective in inhibiting endothelial cell growth than β2GP1 (5 μg/mL ofN-β2GP1 resulted in the same degree of inhibition as 100 μg/mL ofβ2GP1). In the middle panel of FIG. 3, microscopic analysis of thecultures revealed a significant reduction in the number of cells/fieldin the N-β2GP1 containing cultures (FIG. 3 middle panel a). The intactβ2GP1 treated cultures contained a relatively large number of roundpyknotic cells (FIG. 3 middle panel b), and although they were not assparse as the N-β2GP1 cultures, they were much more sparse than thecontrols. Interestingly, other experiments have shown that the effect ofN-β2GP1 on endothelial cells is not species specific. For example, theaddition of N-β2GP1 to subconfluent bovine aorta endothelial cellsresulted in >50% cell death within three days. Intact β2GP1 did not havean effect on these cells. The lack of (species) specificity is notsurprising given the very high homology of β2GP1 between species.

The same experiment outlined for the murine pulmonary microvascularendothelial cells was repeated on B16 melanoma cells to determinewhether the β2GP1-dependent decrease in viable cell number was specificto endothelial cells. As shown in the right panel of FIG. 3, neitherintact β2GP1 nor N-β2GP1 had any effect on the proliferation of tumorcells.

Example 3 β2GP1 Inhibits the Growth of Syngeneic Mouse Tumors

As shown in Example 1, β2GP1 and N-β2GP1 were able to abrogate thegrowth of new vasculature into gel foam implants. Next, experiments werepreformed to determine whether repeated administration of β2GP1 couldalso inhibit vascularization into proliferating syngeneic tumors. Threeindependent mouse models were used to test whether treatment of micewith intact β2GP1 or N-β2GP1 inhibits tumor growth. The first model offibrosarcoma was generated by subcutaneously injecting C3H mice with0.5×10⁶ UV2237 fibrosarcoma cells. When the tumors were palpable (after5 days), buffer, intact β2GP1, or N-β2GP1 was injected subcutaneouslyevery day for two weeks. The dosage of intact β2GP1 or N-β2GP1 fordifferent experiments ranged from 0.1 to 0.5 mg/mouse/day. Thus, thedosage was approximately 5 mg/kg to 25 mg/kg per day. The mice were theninjected intravenously with ⁵¹Cr labeled syngeneic red blood cells todetermine vascular volume (as described in Example 1). The tumors wereexcised, weighed, and assessed for vascularity by determining the amountof labeled red cells present in the tumor tissue (⁵¹Cr) as compared toan aliquot of tail blood. The results presented in FIG. 4 show asignificant reduction in both tumor weight and blood capillary volume inanimals treated with both intact β2GP1 and N-β2GP1.

The second model of B16 melanoma was generated by intravenously andsubcutaneously injecting C57BL/6 mice with 4×10⁵ B16F10 syngeneicmelanoma cells; these injections were designed to model experimentallung metastasis and local tumor growth, respectively. The mice wereinjected intraperitoneally at 24 hour intervals for the entire durationof the experiment (3 weeks) with intact β2GP1 and N-β2GP1 beginning oneday after the tumor cells were injected. Again, the dosage of intactβ2GP1 or N-β2GP1 for different experiments ranged from 0.1 to 0.5mg/mouse/day. The rate of subcutaneous tumor growth was monitored byassessing tumor volume. Lung metastases were counted on day 24. FIG. 5shows a significant reduction in both the size of the subcutaneousgrowing tumors and the number of lung metastasis in animals treated withN-β2GP1.

The third model of Tramp C2RE3 parental orthotopic prostate mode wasgenerated by injecting C57BL/6 mice in the prostate with 0.4×10⁶ TRAMPcells in 40 μl under magnification. On day 3 after the injection,buffer, intact β2GP1 (100 μg/day), or N-β2GP1 (100 μg/day) was injectedintraperitoneally every day. On day 33, the tumors were harvested andweighed. The results of this experiment are shown below in Table 1 (datashown is based on the results of 6 animals/group):

TABLE 1 Effect of β2GP1 on the growth of orthotopically implanted TRAMPcells. Treatment Mean tumor weight (g) SD control 0.388 ± 0.02 intactβ2GP1 0.466 ± 0.11 N-β2GP1 0.147 ± 0.12* *The difference in tumor sizebetween the group treated N-β2GP1 was significantly different from theother two groups (P < 0.05 Student's t-test).

As shown in Table 1, repeated administration of N-β2GP1 resulted in asignificant decrease in tumor size. Taken together, the results of theseexperiments suggest that proteolytically nicked β2GP1 plays an importantregulatory function in angiogenesis by preventing neovascularizationinto syngeneic tumors, as well as into model gel foam assay systems (seeExample 1). Although as shown in FIG. 4, intact β2GP1 was effective ininhibiting the growth of UV2237, it is possible that the subcutaneouslyinjected intact β2GP1 protein undergoes proteolytic cleavage in situthereby generating the active nicked form, which is then able to preventneovascularization into syngeneic tumors. Nevertheless, although N-β2GP1may be the active anti-angiogenic form of the protein, these studies donot rule out the possibility that intact β2GP1 is also active as ananti-angiogenic protein.

Example 4 N-β2GP1 Inhibits Cord Formation, Migration, and Invasion

Since migration and invasion are processes required for the formation ofnew capillaries, β2GP1 was tested in in vitro Matrigel assays for theformation of capillary-like tubes. 96 well plates were first coated withMatrigel (50 μL at 10 mg/mL) according to the manufacturer's protocol.HUVEC (10⁵/mL) were resuspended in medium M-200 containing intact β2GP1or N-β2GP1 (0.1 mg/mL). 200 μl of the cell suspension was then added tothe Matrigel-coated wells. Images were recorded after 4 and 24 hoursincubation at 37° C., as shown in FIG. 6. As shown in the middle panelof FIG. 6, cells plated in the presence of N-β2GP1 seemed to wanderwithout direction on the plate, while control cultures incubated inmedium alone or media supplemented with intact β2GP1 were clearlybeginning to assemble into capillary like structures within ˜4 hours(FIG. 6, top and bottom panels). Differences in the architecture of thefully formed tubes were also evident after 24 hours, although thedifferences were somewhat less striking. Tubes in the N-β2GP1-containingcultures were composed of significantly fewer cells than the controlcultures and gave rise to what appeared to be very thin and fracturedtubes compared to thicker and more robust structures in the controls.

Next, the effect of β2GP1 on endothelial cell migration was examinedusing the 24-well polycarbonate Boyden chambers (8.0 μM pore size,Becton Dickinson). For the migration assay, 750 μL of M-200 mediumcontaining BFGF chemo-attractant was added to the lower chambers of thetranswells. HUVEC (10⁵ cells) were resuspended in 500 μL of M-200 mediumwith or without intact β2GP1 or N-β2GP1, and were added to upperchamber. After incubation at 37° C. for 5 hours, the transwells werefixed and stained, and the number of cells that migrated was counted (10fields/chamber) by two blinded observers. Consistent with the aboveresults for the formation of capillary-like tubes, N-β2GP1 inhibited themigration of endothelial cells in the Boyden chamber invasion assay,while intact β2GP1 had no effect on endothelial cell migration (FIG. 7).Taken together, these data suggest that N-β2GP1 can modulate endothelialcell migration, invasion, and formation of capillary tubes during anangiogenic response.

Example 5 The Effect of β2GP1 on Endothelial Cells In Vivo

In order for a tumor to grow, it must be able to manipulate the hostvasculature to provide an adequate blood supply for its growth. Over thepast decade, many studies have shown that the development of atumor-directed blood supply occurs by several mechanisms that are notnecessarily mutually exclusive. These mechanisms include sproutingangiogenesis, in which host vessels expand into the growing tumor bysprouting from pre-existing vessels (Carmeliet and Jain, Nature407:249-57, 2000), and intussusceptive angiogenesis, which involves therepeated addition of transcapillary pillars in existing tumor vessels,thus allowing the vessels to grow from within themselves (Patan et al.,Microvascular Research 51:260-72, 1996; Burri and Djonov, Mol. Aspects.Med. 23:S1-S27, 2002). New vessels can also be formed by vasculogenesis,in which bone marrow precursors home directly to tumors and generate newvessels in situ (Rafii, J. Clin. Invest. 105:17-9, 2000). In some cases,a tumor can fulfill its needs by co-opting pre-existing vasculaturewithout angiogenesis (Thompson et al., Journal of Pathology 151:323-32,1987; Holash et al., Science 284:1994-8, 1999; Dome et al., Journal ofPathology 197:355-62, 2002). Tumors can also form mosaic vessels wheretumor cells form part of the capillary wall (Chang et al., Proc. Natl.Acad. Sci. (USA) 97:14608-13, 2000). Because of the potentialcontribution of multiple angiogenic mechanisms to tumor growth, growthinhibition by β2GP1 could be due to the inability of a tumor exposed toβ2GP1 to develop an effective blood supply by any one or more of thesemechanisms.

To examine the potential direct effects of β2GP1 on blood vessels, an invivo model system that monitored blood vessel hemodynamics usingreal-time imaging was developed. In this in vivo model system, normalmice were anesthetized and the mesentery was draped on the specimenstage of a dissecting microscope. FIG. 8 shows blood vessel architectureof the normal mesentery. To assess the effects of tumor-derived growthfactors on the architecture of the normal vasculature, vascularendothelial growth factor (VEGF) and nitroglycerin (NG) were applieddirectly to the tissue and recorded under a dissecting microscope.Images were collected in real-time for 1 hour after wetting the area ofinterest with VEGF (0.1 μg/mL; ˜0.05 mL) or NG (40 μg/mL). Consistentwith the known vessel dilation properties of VEGF/VPF and NG, analysisof videotapes revealed what appeared to be increased tortuousness ofvessels and a significant increase in the dilation of small capillariessome of which were barely visible before the addition of VEGF or NG.

Frames of interest were digitized and captured as shown in FIG. 8. Areasof obvious dilation after treatment with VEGF are marked with arrows(FIG. 8, left panels). Note the increased tortuousness of vessels afterthe addition of VEGF, marked by the squiggly trace following somevessels in the VEGF frame (FIG. 8 lower left panel), which was copiedand superimposed on the same vessel in the zero time image (FIG. 8 upperleft panel). Obvious areas of dilated vessels after treatment with NGare also marked, although the magnitude of change is not as great asthat obtained with VEGF. The circled area marks an area where smallvessels became visible after the addition of NG (FIG. 8, right panels).

To better monitor small vessels and the kinetics of VEGF-inducedangioectasia, control mice were injected with 0.1 mg of human serumalbumin 16 hours and 4 hours before they were injected intravenouslywith FITC-HSA. After one hour, the mice were anesthetized and preparedas described above. In many cases, only very few fluorescent vesselscould be seen by fluorescent microscopy in these mice. VEGF was appliedat time zero, the area was videotaped, and frames of interest weredigitized and captured. There are several notable features in thissequence as shown in FIG. 9. First, note the relative sparseness of thefluorescent vessels in the circled area before the addition of VEGF.Within seconds after the addition of VEGF, however, an increasing numberof fluorescent vessels appeared. After 1 minute, the density offluorescent vessels in this area increased significantly, and then beganto gradually disappear over the next 5 minutes. Simultaneously, therewas an increase in diffuse fluorescence throughout the specimen becauseof leakage of the FITC-HSA from the vasculature to the surroundingtissue. Second, note the fluorescent blood vessel marked with the whitearrow before the addition of VEGF (−1 minute). Within 2 minutes afterthe addition of VEGF the fluorescence disappeared. This was because thisvessel quickly filled with red blood cells, the hemoglobin of whichquenches the fluorescence of FITC-HSA. After hemoglobin quenchesfluorescein fluorescence, the vessels are visible only by lightmicroscopy. After 7 minutes, a diffuse area of fluorescence seems toappear which may represent increased vessel permeability and leakage ofsmall plasma proteins (FITC-HSA) to the surrounding tissue. The lightimage taken at 7 minutes also shows a newly visible (red blood cellfilled) blood vessel.

These finding suggests the preexistence of a regulated blood supply,which under normal conditions, is closed. Conceivably, these vesselsopen upon an angiogenic stimulus such as exogenously-supplied VEGF, andalso potentially with endogenously supplied factors from growing tumors.These data raise the possibility that an actively growing tumor recruitsa collateral blood supply from an existing network of closed vesselsreminiscent to “closed blood vessels” of smooth muscle (Krogh A. AContribution to the physiology of the capillaries. Noble Lecture, Dec.11. 1920).

Next, the effect of β2GP1 and N-β2GP1 was examined on this in vivo modelsystem. Mice that were injected intraperitoneally with 0.1 mg of β2GP1or N-β2GP1 16 hours and 4 hours before the experiment were injectedintravenously with FITC-HSA. The mice were then prepared as describedabove, and videotaped throughout the application of VEGF. Interestingly,the opening of vessels (vessel dilation and leakage of FITC-HSA) wascompletely inhibited in animals treated with N-β2GP1, but not in thosetreated intact β2GP1 (FIGS. 10 and 11). To better describe typicalpatterns of fluorescent and “red” vessels, multiple frames fromVEGF-induced angioectasia in mice injected with intact β2GP1, N-β2GP1,and HSA were carefully traced (FIG. 11). Mice injected with intact β2GP1or HSA generally showed a small number of fluorescent vessels thatimmediately increased upon the addition of VEGF and then slowlydisappeared as they filled with red blood cells and became visible underlight microscopy. No fluorescent vessels were seen in animals pretreatedwith N-β2GP1, nor were any apparent differences in the degree of vesseldilation noticed over the time course of the experiment.

To determine if inhibition of angioectasia by N-β2GP1 was specific fortumor-derived factors (e.g., VEGF), the experiments described above wererepeated with nitroglycerin (NG). Mice were injected intraperitoneallywith 0.1 mg of β2GP1, N-β2GP1, or HSA 16 hours and 4 hours before theexperiment. The mice were then prepared as described in FIG. 8, andvideotaped before and after (0.5 hour) the application of NG. Thecircles in FIG. 12 mark areas of obvious vessel dilation in all of theanimals studied. Therefore, unlike the inhibition of VEGF-inducedangioectasia shown above, pretreatment of mice with N-β2GP1 was withouteffect on chemically-induced vessel dilation (FIG. 12). This suggeststhat the inhibitory effect of β2GP1 on blood vessel dilation isindependent of the mitochondrial aldehyde dehydrogenase pathway insmooth muscle cells (Chen et al., Proc. Natl. Acad. Sci. (USA)99:8306-11, 2002).

VEGF was initially purified and identified on the basis of its abilityto induce increased vascular permeability and eventual leak ofintravessel contents (Dvorak et al., Current Topics in Microbiology &Immunology 237:97-132, 1999; Ferrara, Kidney International 56:794-814,1999; Ferrara, Current Topics in Microbiology & Immunology 237:1-30,1999). It was therefore originally coined “vascular permeability factor”(VPF), a characteristic highlighted in the in vivo experiments presentedabove. Blood vessels in many tumors are abnormal in the sense that theyhave numerous fenestrations and openings, defects that make theminherently leaky. Although more studies are needed to assess therelative contribution of angioectasia on tumor growth, the datapresented above raise the possibility that, in addition to trueangiogenesis, angioectasia could provide a significant contribution totumor “vascularity.” If so, inhibition of angiogenesis and angioectasiawith plasmin-cleaved (nicked) β2GP1 might provide aheretofore-unrecognized modality for cancer treatment.

Example 6 Defining the Active Regions of β2GP1

To better understand the therapeutic potential of β2GP1 to inhibit orprevent endothelial cell proliferation, as well as to treatangiogenesis, angioectasia, and tumor growth, it is important to betterdefine the active forms and mechanisms of action of β2GP1. To achievethis goal, expression cloning of domain-deleted β2GP1 will be done todetermine the minimal structural unit of the protein required forinhibition of angiogenesis/angioectasia. Preparative amounts of thesedomain-deleted proteins can be isolated using site-specific enzymaticcleavage. Site-directed mutagenesis of human β2GP1 has clearlydemonstrated that domain V carries the lipid binding region within thelysine-rich sequence motif (281CKNKEKKC288) and a hydrophobic loop(313LAFW316) important to partial intercalation of the protein into thelipid bilayer. Because the anti-angiogenic properties of β2GP1 may bedependent on cleavage at residues Lys 317/Thr 318 which results in asignificant decrease in lipid and membrane binding, it is possible thatthe activity of N-β2GP1 depends on a concomitant alteration in theprotein's structure that might be propagated to one or more domains.

To identify the minimal structural unit of N-β2GP1 responsible for itsanti-angiogenic properties, deletion mutants retaining domain V of theprotein were constructed. The rationale for the design of these mutantsis to specifically delete sequential domains from the N-terminus anddetermine which recombinant protein looses its ability to inhibitendothelial cell proliferation and angiogenesis. For example, if thecritical motif resides in domain III, then Lys 317/Thr 318—cleavedrecombinant proteins D1-5, D2-5 and D3-5 as shown in FIG. 13 should beinhibitory, whereas deletions distal to domain III (D4-5 and D5) wouldnot. Briefly, five cDNA fragments, 979 base pairs (nucleotides 91-1069;D1-5), 790 by (nucleotides 280-1069; D2-5), 625 by (nucleotides445-1069, D3-5), 430 by (nucleotides 640-1069; D4-5) and 253 by(nucleotides 817-1069, D5), were derived by PCR (see FIG. 14) andsubcloned into the pAC 5.1/V5-HisA vector (Invitrogen). The originalβ2GP1 gene used was disclosed in Mehdi et al., Gene 108:293-298, 1991,incorporated herein by reference. The authenticities of the individualclones were determined by sequence analysis. Some of the recombinantproteins have been expressed in the Drosophila S2 expression system(DES, Invitrogen) (see FIG. 15). These recombinant proteins will be usedin experiments such as those outlined in Examples 1-3 to determine whichdomains of the β2GP1 protein inhibit, treat, or prevent endothelial cellproliferation, angiogenesis, angioectasia, and/or tumor growth.

In an effort to rapidly generate large amounts of domain-deleted β2GP1,site-specific enzymatic hydrolysis of intact β2GP1 and intact β2GP1bound to negatively charged lipid vesicles was carried out. The rationalfor generating different domains by this technique is based on ourobservations that β2GP1 undergoes dramatic structural alterations uponbinding to its lipid ligand (Lee et al., Biochim. Biophys. Acta1509:475-84, 2000). To generate the β2GP1 bound to vesicles, β2GP1 (1mg) was incubated with sonicated PS/PC (7/3 mol/mol) vesicles (1 mg/mL)for 2 hours in 10 mM Tris pH 7.1. The precipitated vesicles were washedfree of unbound protein and subsequently incubated with trypsin (1/100)at 37° C. for 20 hours. Native β2GP1 was also incubated with trypsin for20 hours. SDS-PAGE analysis of these digests yielded significantlydifferent profiles (FIG. 16). Two major fragments were generated withlipid-bound β2GP1. A ˜30 kDa fragment (*1) with the N-terminal sequenceDTAVFECLPQH, and a ˜40 kDa (*2) fragment with the N-terminal sequenceYTTFEYPNTIS. These two peptide fragments are consistent with singlepolypeptides encompassing domains III-V and II-V, respectively. Withnative unbound β2GP1, ˜10 kDa (*3) and ˜40 kDa (*4) polypeptides wererecovered. Both fragments had the same GRTCPKPDDLP N-terminus consistentwith polypeptides encompassing domain I and domains I-IV. Since thefragments formed in the presence of lipid contain “protected” domain V,these polypeptides together with the recombinant proteins will be used(before and after plasmin cleavage) to determine which domains of 32GP1are critical to its anti-angiogenic/anti-angioectasia activity.

To determine whether both intact β2GP1 and N-β2GP1 are effective ininhibiting endothelial proliferation, angiogenesis, angioectasia, and/ortumor growth, or alternatively whether N-β2GP1 alone is the activeanti-angiogenic/anti-angioectasia form of the protein, the minimalstructural unit of the protein that is required to inhibitangiogenesis/angioectasia and tumor growth will be determined, in partby using the domain-deleted β2GP1 constructs disclosed above. To helpanswer this question, gel foam implants containing intact β2GP1 will beimplanted into mice as described in Example 1, and removed at 2 dayintervals. After removal, the ratios of intact to nicked protein will bedetermined by amino acid sequence analysis of extracted protein andstaining with monoclonal antibodies specific to intact β2GP1 andN-β2GP1, which can be prepared using methods well known to those ofskill in the art (see, e.g., Horbach et al., Throm. Haemostasis81:87-95, 1999, incorporated herein by reference).

For antibody staining, implants will be removed, frozen in liquid N₂,thin sectioned, and directly stained with fluorescein-labeledanti-(intact)β2GP1 and rhodamine-labeled anti-N-β2GP1. Protein ratioswill be determined by quantitative fluorescence microscopy ratioimaging. For sequence analysis, proteins will be extracted from theimplants with 1% SDS, run on acrylamide gels, and transferred to PDFmembranes. Protein ratios will be determined by the relative ratio ofthe N-terminal sequence of intact β2GP1 (GRTCPKPDDLP) and the N-terminalsequence generated (by protease treatment) at the 317-318 clip site ofN-β2GP1 (TDASDVKPP). Standard β2GP1 will be purified as described (seePolz et al., Int. J. Biochem. 11:265-270, 1980, incorporated herein byreference) and standard N-β2GP1 will be prepared by treatment withplasmin (see Ohkura et al., Blood 91:4173-4179, 1998, incorporatedherein by reference), and purified by HPLC.

Once the active form of the protein is unequivocally determined, theability of the recombinant (plasmin-cleaved) domain-deleted proteins toinhibit angiogenesis into the gel foam implants will also be determined.It is possible that the propensity of the increasingly smallersequential domain-deleted recombinant proteins to diffuse out of theimplants might become problematic. Therefore, diffusion of the proteinsover the time course of the experiment will be monitored by includingtrace amounts of isotopically-labeled (¹²⁵I-) protein. Should thesmaller proteins (˜10 kDa-˜40 kDa) diffuse significantly faster than thefull length protein (˜50 kDa), the fragments will be covalently coupledto albumin using n-hydroxysuccimidyl-human serum albumin, which willeffectively increase fragment size.

To investigate which endothelial cell-dependent pathway may be involvedin inhibiting neovascularization into growing tumors, the following invitro assays can by used to assess the effect of β2GP1, N-β2GP1, andtheir domain-deleted recombinant fragments against human [umbilical cord(HUVEC) and microvascular (HMVEC)] and mouse microvascular endothelialcells. These endothelial cells will include cells from solid tumors ororgans of particular interest (e.g., lungs in the case of B16 melanoma,liver with KM12L4a, and kidney with SN12 and RENCA). A growth inhibitionand apoptosis assay can be done using endothelial cells (1.5×10³) platedin a 96-well plate with 100 μl of medium. After 24 hours (day 0), thetest compounds will be added. Cell growth over 72 hours will bedetermined by staining with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidethiazolylblue (MU assay). Absorbance at 570 nm will be determined with an ELISAreader (Dynatech Laboratories). Duplicate plates can be assessed forapoptosis using the APOPercentage apoptosis assay (Biocolor, BelfastIreland). A tube formation assay can be done using 96-well plates coatedwith ice-cold Matrigel (60 μl of 10 mg/ml; Collaborative Labs) andincubated at 37° C. for 30 min for polymerization. The test compoundsand endothelial cells will be plated on the polymerized matrigel, andafter 24 hours of incubation, triplicate images will be processed. Theeffect of β2GP1 will be compared to untreated controls by measuring tubelength and number of junctions. Finally, a cell migration assay can bedetermine using 48-well Boyden chamber and 8 μm collagen-coatedpolycarbonate filters. The bottom chamber wells will receive mediumalone (baseline) or medium containing a chemo-attractant (bFGF, VEGF).The top chambers receive endothelial cells with or without β2GP1. After5 hours at 37° C., the membrane will be washed, fixed, and stained inDiff-Quick. The filter will be placed on a glass slide with the migratedcells facing down. Negative unstimulated control values will besubtracted from stimulated control and β2GP1-treated values.

Example 7 The Effect of Domain-Deleted Fragments of β2GP1 on EndothelialCells In Vivo

The ability of the domain-deleted recombinant fragments of β2GP1generated in Example 6 to inhibit VEGF-induced angioectasia will bemonitored and recorded in real-time as describe in Example 5. Briefly,mice will be injected with various amounts of N-β2GP1 and itsdomain-deleted fragments at 16 hours and 4 hours before the experiment.The mice will then be injected with fluorescein-albumin. Blood vesseldilation and plasma leak will be monitored in the exposed mesentery bysimultaneous light and fluorescence microscopy immediately before andfollowing the addition of VEGF. Monitoring will be controlled with anelectronically actuated shutter mechanism that switches illuminationfrom a 490 nm interference filtered xenon lamp to a broadband visiblesource at 2 second intervals. By monitoring the rate of closed vessel toplasma flow (green fluorescence) and plasma flow to red cell flow(fluorescein-negative, red visible), vessel dynamics, kinetics and boresize can be accurately quantified.

To assess vascular permeability in these mice, after the mice aretreated with the domain-deleted fragments of β2GP1 as described above,they will be injected intradermally with VEGF, NG, histamine,Angiopoietin −1, or albumin. Vascular permeability will then bedetermined after injecting Evans blue (Thurston et al., Science286:2511-4, 1999, incorporated herein by reference).

Once the minimal inhibitory size of the domain-deleted fragments ofβ2GP1 is determined, synthetic peptides of overlapping sequence will beconstructed and further tested in the studies described above with thegoal of identifying relatively short peptide sequences capable ofinhibiting angioectasia. These studies will likely be very dependent onthe size of the minimal structural unit. For example, if only one or twodomains of N-β2GP1 is required to inhibit angioectasia, constructing aseries of overlapping peptides that encompass the entire polypeptidewould be worthwhile.

Example 8 In Vivo Angiogenesis Assay

The ability of intact β2GP1 and N-β2GP1, as well as intact and nickeddomain deleted mutants of β2GP1, to inhibit angiogenesis will be testedin the gel foam plug assay as described in Example 1 in the presence andabsence of pro-angiogenic molecules. Briefly, 5×5×7 mm pieces of sterilegel foam absorbable sponges (Pharmacia & Upjohn, Peapack, N.J.) will behydrated overnight at 4° C. in phosphate-buffered saline (PBS). Afterremoving excess buffer, 0.4% agarose (100 μl) containing saline(control), the various β2GP1 preparations, and one of the followingproangiogenic molecules: basic fibroblast growth factor (bFGF), vascularendothelial growth factor (VEGF), endothelial growth factor (EGF), tumorgrowth factor-alpha (TGF-α), or platelet-derived growth factor (PDGF)(R&D Systems, Minneapolis, Minn.), will be aliquoted onto each spongecube. After hardening at room temperature, the sponge cubes will beimplanted in mice into subcutaneous pockets formed on both sides of thechest (2-3 cm away from the incision). One gel foam sponge will beinserted into each pocket, and the wound will closed with surgical metalclips. Fourteen days later, the mice will be sacrificed and the gel foamsponges will be recovered, counted for ⁵¹Cr to assess vascular volumeand neovascularization, and finally frozen in OCT compound.

Example 9 Determining the Mechanism by which β2GP1 Inhibits Angiogenesis

A. Inhibition of Growth Factor/Receptor Interactions

Although the results of Examples 1 and 4 clearly suggest that β2GP1inhibits endothelial cell angiogenesis, the results of the in vivo blooddilation studies shown in Example 5 suggest that smooth muscle cellscould be influenced by β2GP1. Thus, other than the possible differentialinteractions of β2GP1 and N-β2GP1 on endothelial cells, β2GP1 mightinhibit the interaction of VEFG with one or more of its receptors onendothelial cells, smooth muscle cells, or both.

The major endothelial cell receptors involved in initiating signaltransduction cascades in response to VEGF is a family of closely relatedreceptors with overlapping specificities known as VEGFR-1 (flt-1),VEGFR-2 (KDR), and VEGFR-3 (flt-3). These receptors bind VEGF-A, whichis also known as vascular permeability factor (VPF), VEGF-B, and VEGF-C,respectively (Veikkola et al., Cancer Res. 60:203-12, 2000; Li andEriksson, International Journal of Biochemistry & Cell Biology 33:421-6,2001). Angiopoietins (Mg's) are another family of endothelial growthfactors that are ligands for the receptor tyrosine kinase, Tie-2 (Daviset al., Cell 87:1161-9, 1996; Yancopoulos et al., Nature 407:242-8,2000). Unlike VEGF, Mg's do not stimulate endothelial cell growth, butinstead likely play a role in blood vessel stabilization. For example,blood vessel leaks induced by VEGF are inhibited by Ang-1 (Thurston etal., Nature Medicine 6:460-3, 2000). Although not wishing to be bound byany particular theory, given the dramatic inhibitory effect of N-β2GP1on VEGF-induced vascular dilation/permeability, N-β2GP1 might act as (i)a VEGF binding protein that shields VEGF from interacting with itsreceptor; (ii) operates as a VEGF-R antagonist; or (iii) an Ang-1agonist (FIG. 20).

To determine whether N-β2GP1 binds to VEGF, VEGF will be incubated with¹²⁵I-labeled intact β2GP1 and N-β2GP, and the ability of specific VEGF(Santa Cruz) and β32GP1 antibodies to co-precipitate both proteins willbe assessed. In the case of anti-VEGF, precipitation of ¹²⁵I will bedetermined. With anti- N-β2GP, the precipitate will be washed, run onSDS-PAGE, transferred to nitrocellulose membranes, and probed withanti-VEGF using conventional techniques. If the results of theseexperiments show that N-β2GP1 does not bind VEGF directly, the followingexperiment will be performed. VEGFR-2 autophosphorylates and becomesactivated upon binding to specific ligands. Endothelial cells will beincubated with VEGF (˜10 ng/mL) in the presence or absence of N-β2GP,and the ratio of phospho-VEGFR (Cell Signaling Technology) to VEGFR(Cascade Biologics) will be determined by quantitativeimmunoprecipitation and/or western blot analysis. Alternatively, cellswill be labeled with ³²P and direct phosphorylation of the receptor willbe determined by immunoprecipitation of ³²P-VEGFR.

If the results of these experiments demonstrate β2GP1-dependentinhibition of phosphorylation, additional experiments will be performedto determine whether N-β2GP1 binds to Tie-1 (agonist) or VEGFR(antagonist). Endothelial cells will be incubated with N-β2GP1 with orwithout VEGF and/or Ang-1. Then the cells will be solubilized innon-ionic detergent and immunoprecipitated with anti-N-β2GP1. FollowingSDS-PAGE, the proteins will be transferred to nitrocellulose membranesand probed with Tie-1 and VEGFR antibodies using conventionaltechniques.

Another approach to identify cell surface epitopes that bind β2GP1 is todirectly crosslink cell bound β2GP1 with its “nearest neighbor” byreaction with hetero- or homo-bifunctional crosslinkers such as BS3.Briefly, unlabeled or radioiodinated-N-β2GP1 will be incubated withendothelial cells (in the presence or absence of VEGF, Ang-1, etc.),washed, and then immobilized with the crosslinking reagent. The cellswill be solubilized, and the β2GP1 complexes will be isolated byaffinity chromatography on anti-β2GP1 columns. If disulfide crosslinkersare used, the linked target epitope will be released with DTT. Proteinswill also be identified by molecular weight, sequence analysis, and bywestern blotting using antibodies against suspect target proteins (e.g.,VEGFR, Ang-1, annexin II).

B. Model Membrane Studies

Several studies have indicated that β2GP1 binds different target ligandsthrough one or more binding sites. For example, both domain V and domainI participate in its interaction with anionic phospholipids (Hong etal., Biochemistry 40:8092-8100, 2001; Hoshino et al., J. Mol. Biol.304:927-939, 2000; Hagihara et al., J. Biochem. 118:129-136, 1995;Hagihara et al., Lupus 4 Suppl 1:S3-S5,1995; Hagihara et al., J.Biochem. 121:128-137, 1997), domain I (Cys-32 through Cys-47) bindscalmodulin (Rojkjaer et al., Biochim. Biophys. Acta 1339:217-225, 1997;Klaerke et al., Biochim. Biophys. Acta 1339:203-216, 1997), and domainIV binds lipid/β2GP1 complex-dependent “antiphospholipid” antibodies.β2GP1 also binds megalin (Moestrup et al., J. Clin. Invest. 102:902-909,1998) and annexin II (Ma et al., J. Biol. Chem. 275:15541-15548, 2000)through as yet unidentified sites. Because the vascular endothelium oftumors express both PS (Ran et al., Cancer Res. 58:4646-4653, 1998) andannexin II (Dreier et al., Histochemistry & Cell Biology 110:137-148,1998; Hajjar et al., J. Biol. Chem. 271:21652-21659, 1996), β2GP1 maybind to endothelial cells through both ligands simultaneously, therebyprotecting the cell from antiangiogenic stimuli that might involvePS/annexin II interactions.

Conceivably N-β2GP1 binds to the cells only through annexin II becauseit cannot bind PS because it is cleaved in domain V (Ohkura et al.,Blood 91:4173-4179, 1998; Hagihara et al., J. Biochem. 118:129-136,1995). Thus, although not wishing to be bound by any particular theory,it is possible that when N-β2GP1 is administered to tumor-bearing mice,it competes with endogenous β2GP1 for binding to annexin II. It ispossible that such a scenario could favor annexin II/PS interactionsthat may lead to signaling pathways (Akiba et al., British Journal ofPharmacology 131:1004-1010, 2000; Bellagamba et al., J. Biol. Chem.272:3195-3199, 1997; Chiang et al., Mol. Cell Biochem. 199:139-147,1999), which results in the induction of PS-induced apoptosis (Uchida etal., J. Biochem. 123:1073-1078, 1998; Miyato et al., FEBS Letters504:73-77, 2001). This hypothetical model of β2GP1-dependent mechanismfor the inhibition of angiogenesis/angioectasia is shown in FIG. 17.

This idea is supported from a thermodynamic standpoint since highaffinity binding of β2GP1 (˜10⁻⁹M) requires “multiple” binding sites(Willems et al., Biochemistry 35:13833-42, 1996) that could be providedby multivalent PS-β2GP1-annexin II interactions. Upon cleavage withplasmin, however, β2GP1 loses its lipid binding capacity and undergoesstructural alterations that could enhance its binding to annexin II.Physiologically, such a scenario could be operative during normal woundhealing through the action of endogenously activated plasmin. From aninterventional standpoint, the affinity of N-β2GP1 for annexin II couldbe significantly greater than that of intact β2GP1. Although stillinconclusive, the results of recent experiments seem to support thishypothesis as shown in FIG. 18.

Vascular endothelial cells were incubated with intact β2GP1 or N-β2GP1for 1 hour at 4° C. The cultures were then washed and stained withfluorescein-conjugated anti-β2GP1. As shown in the left panel of FIG.18, both intact β2GP1 (top) and N-β2GP1 (bottom) bind endothelial cells(left, fluorescence; right: light). Next, ELISA plates were coated withintact β2GP1 or N-β2GP1. The plates were blocked with 1% ovalbumin andbiotin labeled annexin was added. After 1 hour, the plates were washedand developed with peroxidase-avidin. As shown in the middle panel ofFIG. 18, both intact β2GP1 and N-β2GP1 bind annexin II. Finally, ELISAplates were coated with phosphatidylserine, blocked with 1% ovalbumin,and incubated with intact β2GP1 or N-β2GP1 preparations. Protein bindingto the plates was determined with rabbit β2GP1 antibodies followed byperoxidase-conjugated anti-rabbit lg. As shown in the right panel ofFIG. 18, only intact β2GP1 binds lipid, with a comparatively significantdecrease in the binding of N-β2GP1.

To determine if N-β2GP1 could compete for the binding of intact β2GP1,both intact β2GP1 and N-β2GP1 were labeled with ¹²⁵I (1 mCi/100 μgprotein with iodogen), and added in the presence of increasingconcentrations of unlabeled nicked protein to HUVECS endothelial cellsin 24 well plates (100 μg/mL by dilution with unlabeled protein). Theplates were washed after 1 hour, and uptake was determined byscintillation counting. As shown in FIG. 19, increasing concentrationsof unlabeled N-β2GP1 competed for the binding of both ¹²⁵I-labeledproteins, suggesting that both intact β2GP1 and N-β2GP1 bind to the sameendothelial cell binding site. This finding is particularly importantsince it suggests that the nicked protein could displace intact β2GP1,regardless of its ability to bind lipid.

The following are additional experiments that will be used to test thehypothesis that N-β2GP1 induces apoptosis through a PS/annexinII-dependent mechanism. These experiments will directly determine theassociation of intact β2GP1 and N-β2GP1 with PS and with annexin II, aswell as the association of PS directly to annexin in both cell freemodel membrane systems and in in vitro cultivated endothelial cells.First, resonance energy transfer (RET), as described in Lee et al.,Biochim. Biophys. Acta 1509:475, 2000, incorporated herein by reference,can be used to detect multiple specific β2GP1 interactions with lipid inmodel membranes. These experiments can be used to specifically test theassociation of intact β2GP1 and N-β2GP1 with PS and with annexin II, andthe concomitant formation of PS/β2GP1 and PS/annexin II complexes,respectively.

Briefly, phosphatidylcholine vesicles containing increasing amounts ofNBD-labeled PS or control NBD-labeled PC (the energy donors) will begenerated by sonication or ethanol injection. The interaction of thelabeled PS will then be monitored by incubating the vesicles withrhodamine-labeled intact β2GP1 and N-β2GP1. The kinetics of theinteraction will then be monitored in real time. Similar experimentswill be done to monitor the ability of the NBD-lipid to directlyassociate with rhodamine labeled annexin II tetramers intercalated intothe vesicle membrane (Raynor et al., Biochemistry 38:5089-5095, 1999;Singh and Liu, Arch. Biochem. Biophys. 381:235-240, 2000; incorporatedherein by reference) in the presence of intact β2GP1 and N-β2GP1.

Ellipsometric measurements of protein adsorption to lipid bilayers canalso be used determine dissociation constants for annexin II toimmobilized intact β2GP1 and N-β2GP1, essentially as previouslydescribed for annexin V (Balasubramanian et al., Biochemistry40:8672-8676, 2001, incorporated herein by reference). The role of lipidwill also be determined by depositing planar lipid bilayers on siliconslides by immersion for 5 minutes in a stirred suspension of sonicatedvesicles in Tris buffer containing appropriate concentrations of PS.Protein adsorption to the lipid bilayers will be assessed using β2GP1,N-β2GP1, and annexin H alone. Additive protein adsorptions can also bedetermined.

The binding of PS to targets in the endothelial cell membrane will alsobe assessed using iodinated photoactivatable lipid analogs as disclosedin Schroit et al., Biochemistry 26:1812-1819, 1987; Schroit et al.,Biochemistry 29:10303-10306, 1990; Connor et al., J. Biol. Chem.267:19412-19417, 1992; and Connor and Schroit, Biochemistry28:9680-9685, 1989, incorporated herein by reference. ¹²⁵I-labeled-N₃-PSand control ¹²⁵I-labeled-N₃-PC can be exchanged into endothelial cellsat 4° C. β2GP1 and N-β2GP1 will be incubated with the cells for 30minutes. The cells will then be photolysed, solubilized, and analyzed bySDS-PAGE. Proteins labeled with lipid probes will be detected byautoradiograhy and identified by molecular weight, sequence analysis,and western blotting to suspect target ligands.

Example 10 Therapeutic Potential of β2GP1

Additional experiments may be performed to determine optimal route,dosage, and frequency of β2GP1 administration for maximal inhibition oftumor growth. Additionally, the minimal effective structural unit ofβ2GP1 identified in the in vitro assays described above (smallestrecombinant domain-deleted fragment) can be tested for efficacy. Oneexperimental system that models colon cancer is described as follows:mice will be anesthetized, washed, and a left-center subcostal incisionwill be made to expose the spleen. The spleen is lifted onto sterilegauze just outside of the wound and 0.05 ml of the cell suspension (10⁶cultured KM12L4a) will be injected into the parenchyma. The incisionwill be closed in one layer with surgical clips. The mice are randomizedinto treatment groups (10 mice/group) on about day 7. Therapy willconsist of either 5-fluorouracil alone (50 mg/kg, 3×/week for 4 weeks);gemcitabine (125 mg/kg, twice weekly for 4 weeks), and β2GP1 and N-β2GP1alone, or in combination with the chemotherapeutic agents. When micebecome symptomatic of liver tumor burden (ascites, palpable tumor,posture, body weight), they will be sacrificed and the spleens, livers,and adjacent lymph nodes will be removed for determination of tumorburden and for immunohistochemistry.

Another experimental model that may be used is primary and metastaticrenal cell carcinoma of human cancer in nude mice. In this protocol,nude mice are washed with betadine and a left subcostal incision is madeto expose the left kidney. A 30-gauge needle is inserted from the lowerpole to just below the renal subcapsule on the superior pole of thekidney. A 0.05 ml cell suspension (10⁶ cultured SN12pm6 cells) isinjected, resulting in a subcapsular bleb. The incision is closed in onelayer with wound clips. Nephrectomy of the injected kidney is performedon about day 30. At the time of nephrectomy, an incision parallel to theoriginal is made to expose the tumorous left kidney, the kidney isremoved by ligature below the organ pedicle, and the wound is closedwith clips. This protocol results in a high incidence of spontaneouslung metastasis of moderate tumor burden. Some groups of mice (10mice/group) will be treated for the growth of primary tumor with intactβ2GP1, N-β2GP1, or Taxol, 100 μg/dose, 1×/week, for 4 weeks, or acombination thereof. The therapeutic efficacy will be determined byweight of the primary tumors (kidneys). Other groups of mice will betreated after nephrectomy to evaluate the effects of single agent orcombination therapy on the development of spontaneous lung metastasisthat develop about 3-4 weeks post-nephrectomy. The lungs of mice will beharvested and the number and size of individual lung tumor nodulesdetermined.

The model described above for human renal cell carcinoma can also beevaluated in a syngeneic murine model of renal adenocarcinoma (RENCA) inBalb/c mice. The primary tumors are injected as described above (10⁵cultured RENCA cells) and nephrectomy occurs on about day 14. The lungsof mice will be harvested on about day 35 to determine the extent ofspontaneous lung metastasis.

In another model, the effect of β2GP1 and N-β2GP1 on subcutaneous growthof A237 human melanoma cells in nude mice can be assessed. In thisprotocol, nude mice will receive a subcutaneous injection of 10⁶cultured A375 cells on day 0 and therapy will begin when the tumors areabout 2-4 mm in diameter. Treatment groups (10 mice/group) arerandomized to receive either doxorubicin (10 mg/kg, 1× week for twoweeks), β2GP1, N-β2GP1, or a combination thereof. Tumor growth can beassayed by two perpendicular measurements of tumor diameter (estimatedtumor volume is a x b²/2, where a and b are the long and shortdiameters, respectively).

In yet another model, the effect of β2GP1 and N-β2GP1 on a model ofexperimental lung metastasis of B16 melanoma in syngeneic C57BL/6 micecan be determined. In this protocol, mice receive an intravenousinjection of 40,000 cultured B16F10 cells on day 0 and therapy begins onabout day 4. Using 10 mice/group, mice receive either doxorubicin (10mg/kg, 1×/week for two weeks), β2GP1, N-β2GP1, or a combination thereof.On day 21, mice are sacrificed and the number of individual lung tumornodules are determined. Necropsy of mice will determine the extent ofother organ metastases and harvest of tissue for immunohistochemistryfor the determination of blood vessel cell density and cellularapoptosis.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are chemically or physiologicallyrelated may be substituted for the agents described herein while thesame or similar results would be achieved. All such similar substitutesand modifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

1. A method of inhibiting angiogenesis within a tissue at a site in asubject having an angiogenic disease, comprising administering aneffective amount of a β2GP1 polypeptide to cells associated with thetissue in said subject, wherein said β2GP1 polypeptide is β2GP1 domainI, β2GP1 domain II, β2GP1 domain III, β2GP1 domain IV, β2GP1 domain V,β2GP1 domains I through IV, β2GP1 domains II through V, β2GP1 domainsIII through V, or β2GP1 domains IV and V, and wherein the amount iseffective to inhibit angiogenesis within the tissue.
 2. The method ofclaim 1, wherein said β2GP1 polypeptide is β2GP1 domain I.
 3. The methodof claim 1, wherein said β2GP1 polypeptide is β2GP1 domain II.
 4. Themethod of claim 1, wherein the β2GP1 polypeptide is β2GP1 domain III. 5.The method of claim 1, wherein the β2GP1 polypeptide is β2GP1 domain IV.6. The method of claim 1, wherein the β32GP1 polypeptide is β2GP1 domainV.
 7. The method of claim 1, wherein the cells associated with thetissue are vascular endothelial cells.
 8. (canceled)
 9. The method ofclaim 1, wherein the tissue is a tumor.
 10. The method of claim 9,wherein said method further inhibits neovascularization into the tumor.11. The method of claim 1, further comprising administering anantiangiogenic agent to the cells in conjunction with said β2GP1polypeptide.
 12. The method of claim 11, wherein the antiangiogenicagent is selected from the group consisting of angiostatin, endostatin,trastuzumab, thrombospondin, IFN-α, TIMP-1, PF4, fumagillin, a tyrosinekinase inhibitor, an antibody to VEGF, and mixtures thereof.
 13. Themethod of claim 45, wherein the β2GP1 polypeptide is supplied to the eyetopically.
 14. A method of inhibiting endothelial cell proliferation,comprising administering an effective amount of a β2GP1 polypeptide tothe endothelial cells, wherein said β2GP1 polypeptide is β2GP1 domain I,β2GP1 domain II, β2GP1 domain III, β2GP1 domain IV, β2GP1 domain V,β2GP1 domains I through IV, β2GP1 domains II through V, β2GP1 domainsIII through V, or β2GP1 domains IV and V, and wherein the amount iseffective to inhibit endothelial cell proliferation.
 15. (canceled) 16.(canceled)
 17. The method of claim 14, wherein said method furtherinhibits endothelial cell migration.
 18. The method of claim 14, whereinsaid method further inhibits endothelial cell differentiation.
 19. Themethod of claim 18, wherein the endothelial cells are inhibited fromdifferentiating into tubular capillary structures.
 20. The method ofclaim 1, wherein the tissue is a neoplasm.
 21. The method of claim 1,wherein said β2GP1 polypeptide is β2GP1 domains I through IV.
 22. Themethod of claim 1, wherein said β2GP1 polypeptide is β2GP1 domains IIthrough V.
 23. The method of claim 1, wherein the β2GP1 polypeptide isβ2GP1 domains III through V.
 24. The method of claim 1, wherein theβ2GP1 polypeptide is β2GP1 domains IV and V.
 25. The method of claim 9,wherein said method further inhibits metastasis of the tumor. 26.-29.(canceled)
 30. The method of claim 9, further comprising administering atherapeutic agent useful in the treatment of the tumor in conjunctionwith said β2GP1 polypeptide.
 31. The method of claim 30, wherein thetherapeutic agent is selected from the group consisting of cisplatin,doxorubicin, paclitaxel, vincristine, tamoxifen, taxotere, methotrexate,carboplatin and vinblastine. 32.-34. (canceled)
 35. The method of claim1, wherein the subject is human.
 36. The method of claim 1, wherein theβ2GP1 polypeptide is β2GP1 domain V, β2GP1 domains II through V, β2GP1domains III through V, or β2GP1 domains IV and V.
 37. The method ofclaim 36, wherein the β2GP1 domain V is nicked β2GP1 domain V (N-β2GP1domain V).
 38. The method of claim 1, wherein the route ofadministration to the subject is oral, intravenous, intramuscular,intrathecal, intradermal, intraperitoneal, subcutaneous, intrapleural,intrauterine, rectal, vaginal, intratumor, transdermal, or transmucosal.39.-43. (canceled)
 44. The method of claim 1, wherein said angiogenicdisease is an angiogenic disease of the eve, skin, joints,gastrointestinal tract, reproductive system, or involved with fractureor wound healing.
 45. The method of claim 44, wherein said angiogenicdisease is an angiogenic disease of the eye.
 46. The method of claim 44,wherein said angiogenic disease is an angiogenic disease of the skin.47. The method of claim 44, wherein said angiogenic disease is anangiogenic disease of the joints.
 48. The method of claim 44, whereinthe angiogenic disease is an angiogenic disease of the gastrointestinaltract.
 49. The method of claim 45, wherein the angiogenic disease is acorneal disease, hypoxia, infection, diabetic retinopathy, retrolentalfibroplasia, trachoma, neovascular glaucoma, rubeosis, maculardegeneration or an angiogenic disease associated with an eye injury orlaser surgery.
 50. The method of claim 1, wherein the site is dermis,epidermis, endometrium, retina, surgical wound, gastrointestinal tract,umbilical cord, liver, kidney, reproductive system, lymphoid system,central nervous system, breast tissue, urinary tract, bone, muscle, orrespiratory tract.
 51. A pharmaceutical composition comprising a β2GP1polypeptide and a second antiangiogenic agent useful for the inhibitionof angiogenesis; wherein said β2GP1 polypeptide is β2GP1 domain I, β2GP1domain II, β2GP1 domain III, β2GP1 domain IV, β2GP1 domain V, β2GP1domains I through IV, β2GP1 domains II through V, 02GP1 domains IIIthrough V, or β2GP1 domains IV and V.
 52. (canceled)
 53. (canceled) 54.The composition of claim 51, wherein the second antiangiogenic agent isselected from the group consisting of angiostatin, endostatin,trastuzumab, thrombospondin, IFN-α, TIMP-1, PF4, a tyrosine kinaseinhibitor, an antibody to VEGF, and fumagillin.
 55. A pharmaceuticalcomposition comprising a β2GP1 polypeptide and a second therapeuticagent; wherein said β2GP1 polypeptide is β2GP1 domain I, β2GP1 domainII, β2GP1 domain III, β2GP1 domain IV, β2GP1 domain V, β2GP1 domains Ithrough IV, β2GP1 domains II through V, β2GP1 domains III through V, orβ2GP1 domains IV and V, and wherein said second therapeutic agent isuseful for the treatment of a neoplasm.
 56. (canceled)
 57. (canceled)58. The composition of claim 55, wherein the second therapeutic agent isselected from the group consisting of cisplatin, doxorubicin,paclitaxel, vincristine tamoxifen, taxotere, methotrexate, carboplatinand vinblastine.
 59. The method of claim 46, wherein the angiogenicdisease is psoriasis, scleroderma, neovascularization as a consequenceof infection, cat scratch disease, bacterial ulceration, lupuserythematosus, telangiectasia, or hypertrophic scars.
 60. The method ofclaim 47, wherein the angiogenic disease is arthritis, rheumatoidarthritis, hemophiliac joints, lupus erythematosus, immune-inflammation,or non-immune inflammation.
 61. The method of claim 48, wherein theangiogenic disease is angioectasia, telangiectasia, intestinaladhesions, Crohn's disease, Oster-Webber Syndrome, or peptic ulcer. 62.The method of claim 44, wherein the angiogenic disease is an angiogenicdisease associated with a fracture or would healing.
 63. The method ofclaim 62, wherein the angiogenic disease is associated with excessivewound repair, wound granularization, or an ischemic limb.